SS3) 

i 


UNIVERSITY    OF    CALIFORNIA    PUBLICATIONS 
-^IN  - 

ZOOLOGY 

Vol.  13,  No.  4,  pp.  43-122,  pis.  3-7,  4  text  figures  May  4,  1914 


DIPLODINIUM  ECAUDATUM 

WITH  AN  ACCOUNT  OF  ITS  NEUROMOTOR 
APPARATUS 


BY 
ROBERT   G.   SHARP 


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age of  Strix  occidentals  occidentals  (Xantus),  by  Harry  S.  Swarth. 

Pp.  1-8.    May,  1910      10 

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4.  The  Linnet  of  the  Hawaiian  Islands:  a  Problem  in  Speciation,  by 

Joseph  Grinnell.    Pp.  179-195.    February,  1911  15 

5.  The  Modesto  Song  Sparrow,  by  Joseph  GrinneU.    Pp.  197-199.    Feb- 

ruary, 1911  03 

6.  Two  New  Species  of  Marmots  from  Northwestern  America,  by  H.  8. 

Swarth.    Pp.  201-204.    February,  1911  Ofl 

7.  Mammals  of  the  Alexander  Nevada  Expedition  of  1909,  by  Walter  P. 

Taylor.    Pp.  205-307.    June,  1911  1.0(1 

8.  Description  of  a  New  Spotted  Towhee  from  the  Great  Basin,  by  J. 

Grinnell.    Pp.  309-5*11.    August,  1911  _.      .05 

9.  Description  of  a  New  Hairy  Woodpecker  from  Southeastern  Alaska,  by 

H.  S.  Swarth.    Pp.  313-318.    October,  1911  „      .05 

10.  Field  Notes  on  Amphibians,  Reptiles  and  Birds  of  Northern  Humboldt 
County,  Nevada,  with  a  Discussion  of  Some  of  the  Fauna!  Features 
of  the  Begion,  by  Walter  P.  Taylor.  Pp.  319-436,  plates  7-12. 

February,  1912 —    1.00 

Index,  pp.  437-446. 

VoL  8.      1.  The  Vertical  Distribution  of  Eumlanus  elongatus  in  the  San  Diego 

Eegion  during  1909,  by  Calvin  O.  Esterly.    Pp.  1-7.    May,  1911 .10 

2.  New  and  Eare  Fishes  from  Southern  California,  by  Edwin  Chapin 

Starks  and  William  M.  Mann.    Pp.  9-19,  2  text-figures.    July,  1911.      .10 

3.  Classification  and  Vertical  Distribution  of  the  Chaetognatha  of  the  Sam 

Diego  Eegion,  Including  Eedescriptions  of  Some  Doubtful  Species  of 

the  Group,  by  Ellis  L.  Michael.  Pp.  21-186,  pis.  1-8.  December,  1911.    1.7fl 

4.  Dinoflagellata  of  the  San  Diego  Eegion,  IV.  The  Genus  Gonyaulax,  with 

Notes  on  Its  Skeletal  Morphology  and  a  Discussion  of  Its  Generic 
and  Specific  Characters,  by  Charles  Atwood  Kofoid.  Pp.  187-286, 
plates  9-17. 


UNIVERSITY    OF    CALIFORNIA    PUBLICATIONS 

IN 

ZOOLOGY 

Vol.  13,  No.  4,  pp.  43-122,  pis.  3-7,  4  text  figures  May  4,  1914 


DIPLODINIUM  ECAUDATUM 

WITH   AN   ACCOUNT    OF    ITS   NEUROMOTOR 
APPARATUS 


BY 

EGBERT  G.   SHARP 


CONTENTS 

PAGE 

A.  Introduction    44 

1.  Review  of  literature 45 

2.  Present  status  49 

3.  Validity  of  species  51 

B.  Technique 52 

1.  Living  material  52 

2.  Methods  of  fixation  and  staining  55 

C.  General  discussion  of  the  genus  58 

1.  Diplodinium  ecaudatum  forma  ecaudatum  62 

(a)   Ectoplasmic  structures  63 

(1)  Cuticle 63 

(2)  Skeletal  areas 64 

(3)  Ectoplasm    65 

(4)  Skeletal  structures 65 

(5)  Boundary  layer  67 

Entoplasm  68 

Organs  of  the  body  69 

(1)  Macronucleus    69 

(2)  Micronucleus  71 

(3)  Organs  of  locomotion  72 

(4)  Organs  of  food-taking 77 

(5)  Organs  of  defecation  80 

(6)  Organs  of  excretion  82 

(7)  Neuromotor  apparatus  82 

(d)   Retracted  form  88 

2.  Diplodinium  ecaudatum  forma  caudatum  90 

3.  Djiplodiuium  ecaudatum  forma  bicaudatum  forma  nova  92 


•'  * 


44  University  of  California  Publications  in  Zoology        [VOL.  13 

PAGE 

4.  Diplodinium  ecaudatum  forma  tricaudatum  forma  nova  ........................     92 

5.  Diplodinium  ecaudatum  forma  quadricaudatum  forma  nova  ................     93 

6.  Diplodinium  eeaudatum  forma  cattanei  ....................................................     94 

7.  Table  of  dimensions  ........................................................................................     95 

8.  Observations  on  the  living  material  ..........................................................     95 

D.  Conclusions   ..............................................................................................................  101 

E.  Addendum.     Discussion  of  Braune's  paper  ......................................................  103 

F.  Bibliography  ............................................................................................................  110 


INTRODUCTION 

During  the  winter  of  1909  Professor  C.  A.  Kofoid,  of  the 
University  of  California,  called  my  attention  to  the  papers  of  Fioren- 
tini  (1889)  and  Eberlein  (1895)  dealing  with  the  protozoan  fauna 
of  the  stomachs  of  ruminants,  and  pointed  out  the  fact  that  the  life- 
history  of  these  protozoans  had  not  yet  been  traced.  This  appealed 
to  me  as  an  interesting  bit  of  research,  and  acting  upon  Professor 
Kofoid 's  further  suggestion  I  first  made  certain  that  material  was 
procurable  and  then  consulted  the  literature  upon  the  subject.  The 
papers  of  Schuberg  (1888),  Fiorentini  (1889),  Eberlein  (1895),  and 
Giinther  (1899,  1900),  were  at  hand  and  from  a  hasty  perusal  of  these 
it  became  evident  that  the  genus  Diplodinium  offered  the  greater  pos- 
sibilities as  a  research  problem.  Most  of  the  described  species  of  this 
genus  were  present  in  the  stomach  fluid  which  I  was  able  to  obtain,  and 
especially  abundant  were  the  species  which  Fiorentini  (1889)  had 
described  as  Diplodinium  ecaudatum,  D.  caudatum,  and  D.  cattanei. 

As  my  observations  proceeded  it  became  evident  that  the  Protozoa 
infecting  the  stomachs  of  western  cattle  presented  many  differences 
in  structure  from  those  figured  and  described  both  by  Fiorentini 
(1889)  and  by  Eberlein  (1895).  This  was  found  to  be  especially  true 
in  the  case  of  those  species  described  as  belonging  to  the  genus 
Diplodinium. 

The  present  paper  deals  with  the  morphology  of  Diplodinium 
ecaudatum  Fiorentini  including  D.  caudatum  Fiorentini,  and  D.  cat- 
tanei Fiorentini,  together  with  a  description  of  three  new  forms  of  this 
species,  viz.,  Diplodinium  ecaudatum  forma  bicaudatum,  Diplodinium 
ecaudatum  forma  tricaudatum,  and  Diplodinium  ecaudatum  forma 
quadricaudatum,  all  of  which  are  found  in  the  first  and  second  divisions 
of  the  stomach  of  western  cattle. 


1914]  Sharp:  Diplodinium  ecaudatum  45 

ACKNOWLEDGMENTS 

Whatever  there  be  of  merit  in  the  methods  used  and  the  results 
so  far  obtained  is  due  to  the  kindly  and  helpful  suggestions  and 
interest  of  Professor  Kofoid,  under  whose  direction  the  work  has 
been  done. 

My  acknowledgments  are  also  due  to  Mr.  R.  B.  Brown,  Superin- 
tendent of  the  Oakland  Meat  and  Packing  Company,  for  his  personal 
interest  and  assistance  in  procuring  material  for  this  study  in  the 
most  advantageous  manner. 

LITERATURE 

The  literature  upon  this  subject  is  not  extensive,  although  covering 
a  period  of  more  than  seventy  years.  A  brief  review  follows. 

The  first  information  regarding  the  presence  of  protozoans  in  the 
stomach  of  the  ox  was  given  by  two  French  scientists,  Grube  and 
Delafond  (1843).  In  this  communication  they  presented  a  short 
general  account  of  the  stomach  parasites  of  the  horse,  the  dog,  and 
the  pig,  as  well  as  those  of  ruminants.  This  work  is  important  because 
of  the  early  date  at  which  it  was  done  and  because  it  opened  up  a 
new  field  of  investigation.  The  results,  according  to  our  modern 
conception,  were  by  no  means  accurate,  and  the  work,  although  carried 
out  with  great  skill,  considering  the  limited  means  of  that  period, 
is  unfortunately  unaccompanied  by  illustrations ;  a  fact  which  makes 
it  difficult  to  determine  which  protozoans  the  investigators  observed. 
In  the  case  of  the  ox  they  gave  descriptions  of  four  species,  from 
which  it  is  almost  certain  that  they  had  observed  those  protozoans  now 
classified  as  belonging  to  the  genera  Ophryoscolex,  Entodinium, 
Diplodinium  and  Isotricha.  Even  at  this  early  date  (1843)  they 
called  attention  to  the  fact  that  in  the  ox  these  protozoans  occur  in  the 
living  condition  only  in  the  first  two  subdivisions  of  the  stomach, 
the  rumen  and  the  reticulum;  while  in  the  third  and  fourth  sub- 
divisions, the  omasum  and  the  abomasum,  only  dead  and  disintegrated 
animals  are  found. 

The  next  information  regarding  these  protozoans  was  given  by 
Colin  (1854).  In  a  discussion  of  the  digestive  processes  which  take 
place  in  the  stomachs  of  the  ruminants  Colin  reproduces  eighteen  draw- 
ings, but  gives  only  brief  descriptions.  The  figures  demonstrate 
clearly,  however,  that  Colin  saw  and  recognized  species  subsequently 
described  in  the  genera  Diplodinium,  Entodinium  and  Isotricha. 


46  University  of  California  Publications  in  Zoology        [VOL.  13 

Stein  (1858,  1859,  1861,  and  1867)  followed  with  short  but  excel- 
lent descriptions.  Although  lacking  illustrations,  Stein's  work  was 
good;  he  gave  model  descriptions  and  a  scientific  classification.  He 
described  the  genera  Ophryoscolex,  Entodinium  and  Isotricha.  In 
1861  he  added  to  his  earlier  work,  and  again  in  1867.  In  this  later 
work  he  classifies  for  the  first  time  the  genera  Ophryoscolex  and 
Entodinium  under  the  family  name  Ophryoscolecidae. 

In  1869  Weiss  confirmed  the  presence  of  these  infusorians  in  the 
stomachs  of  ruminants  and  contented  himself  principally  with  a  report 
upon  the  writings  of  Delafond  and  Stein. 

Leuckart  (1879-1886)  only  reviewed  the  researches  of  Stein. 

In  1872  Ziirn  did  a  large  amount  of  work,  but  owing  to  poor 
technique  his  material  was  bad,  and  consequently  his  descriptions 
were  faulty  and  his  figures  inaccurate.  In  a  second  edition  of  his 
work  (Ziirn  and  Plaut,  1887-1889),  he  abandoned  his  earlier  figures 
and  enlarged  upon  his  descriptions  by  quoting  from  Schuberg's  (1888) 
discoveries. 

Kent  (1881)  published,  in  his  Manual  of  the  Infusoria,  a  com- 
pilation of  the  work  of  Stein  (1858,  1859,  and  1861),  but  this  com- 
pilation contained  many  errors.  Kent,  as  a  matter  of  fact,  added 
nothing  along  this  line  to  the  work  of  his  predecessors. 

List  (1885)  gave  little  that  was  new.  The  animals  over  which  he 
worked  were  either  dead  or  had  been  affected  by  the  water.  His 
work,  in  so  far  as  it  relates  to  the  ciliates,  is  without  present  value. 

In  1888  Schuberg  published  the  results  of  his  work  on  Buetschlia, 
Isotricha,  Dasytricha,  and  Entodinium.  His  work  wras  the  most  scien- 
tific and  most  complete  done  up  to  his  time  and  in  some  respects  is 
still  the  best.  He  described  two  new  genera,  which  he  named 
Buetschlia  (with  two  species)  and  Dasytricha  (with  one  species).  He 
added  several  species  to  Stein's  genus  Entodinium  and  divided  the 
genus  into  two  genera,  i.e.,  Entodinium  and  Diplodinium,  although  he 
gives  neither  a  description  nor  an  illustration  of  the  genus  Diplo- 
dinium— in  fact  nothing  beyond  the  mere  statement  that  the  genus 
Diplodinium  is  provided  with  two  sets  of  membranelles,  one  around 
the  mouth  opening  and  the  other  on  the  dorsal  side.  His  methods 
he  describes  in  detail  and  to  these  we  shall  have  occasion  to  refer 
later.  He  intended  to  write  a  second  lengthy  paper,  but  never,  so 
far  as  I  am  able  to  determine,  was  this  published.  In  1891  he  pub- 
lished a  short  paper,  which  contains  no  illustrations  and  is  occupied 
largely  with  a  description  of  some  of  the  structural  relationships  and 


Sharp:  Diplodinium  ecaudatum  47 

a  partial  description  of  the  process  of  division  in  the  Ophryoscole- 
cidae  and  in  Dasytricha. 

Fiorentini  (1889)  published  a  short  paper  dealing  mainly  with 
the  genera  Diplodinium  and  Entodinium.  This  paper  is  profusely 
illustrated  and  although  the  drawings  are  crude  as  compared  to  those 
of  either  Schuberg  or  of  Eberlein,  they  are  still,  in  some  respects,  more 
true  to  life  than  are  those  of  either  of  the  others.  Fiorentini  is 
apparently  the  first  to  describe  individual  species  of  the  genus 
Diplodinium.  He  figures  and  briefly  describes  nine  species  (see  table 
below).  To  the  genus  Entodinium  he  adds  two  new  species  and  to 
the  genus  Buetscklia  one  new  species  (see  table  below) .  It  seems  that 
Fiorentini  contented  himself  with  the  discovery  and  naming  of  new 
species  rather  than  with  the  careful  and  accurate  description  and 
illustration  of  the  species  upon  which  he  worked.  In  general  his 
methods  were  crude  and  his  descriptions  too  brief.  To  these  we  shall 
refer  again. 

It  is  interesting  to  note  in  this  connection  that  Fiorentini  (1890) 
in  his  paper  dealing  with  the  Protozoa  parasitic  in  the  intestinal 
tract  of  the  horse,  describes  two  new  species  which  he  refers  to  the 
genus  Entodinium  Stein,  i.e.,  "Entodinium  valvatum"  and  "Ento- 
dinium bipalmatum,"  and  also  two  new  species  which  he  adds  to  the 
genus  Diplodinium  Schuberg,  i.e.,  "Diplodinium  uncinatum"  and 
"Diplodinium  unifasciculatum."  Since,  however,  the  descriptions  of 
these  new  species  are  not  in  accord  with  the  characteristics  of  the 
genera  Entodinium  and  Diplodinium  respectively  as  laid  down  by 
Stein  (1858,  p.  69),  and  Schuberg  (1888,  p.  404),  and  Biitschli 
(1888,  p.  1783),  it  is  evident  that  Fiorentini  made  a  serious  mistake 
in  assigning  these  species  to  the  genera  Entodinium  and  Diplodinium, 
a  fact  which  was  very  clearly  pointed  out  by  Bundle  (1895,  pp.  296- 
298  and  309-312).  In  this  paper  Bundle  founds  the  new  genus 
Cycloposthium  to  which  he  assigns  Entodinium  bipalmatum  Fioren- 
tini, which  therefore  properly  becomes  Cycloposthium  bipalmatum 
(Fiorentini)  and  stands  as  the  type  species.  In  the  same  paper 
Bundle  (1895)  founded  another  new  genus,  Blepharocorys,  to  which 
he  referred  both  the  Entodinium  valvatum  Fiorentini  which  then 
becomes  Blepharocorys  valvatum  (Fiorentini)  type  species,  and  also 
Diplodinium  uncinatum  Fiorentini  which,  therefore,  becomes  Blephar- 
ocorys uncinatum  (Fiorentini).  Bundle  (1895)  does  not  discuss 
Diplodinium  unifasciculatum,  but  it  can  be  seen  at  a  glance  that  an 
animal  such  as  Fiorentini  has  pictured  as  Diplodinium  unifasci- 


48  University  of  California  Publications  in  Zoology        [VOL.  13 

culatum  is  not  correctly  referable  either  to  the  genus  Diplodinium  or 
to  Entodinium.  In  my  opinion  it  may  be  referred  to  the  genus 
Blepharocorys  Bundle. 

The  next  work  of  importance  is  that  of  Eberlein  (1895),  who 
published  a  rather  voluminous  account  of  his  investigations  as  well  as 
a  resume  of  all  that  had  been  done  in  this  field  by  previous  investi- 
gators. His  methods  were  much  superior  to  those  of  his  predecessors 
and  his  work  as  a  whole  bears  the  stamp  of  thoroughness,  complete- 
ness, and  scientific  accuracy.  He  adds  one  new  species,  Ophryoscolex 
caudatus,  and  claims  the  discovery  of  another,  which  he  names 
Diplodinium  caudatum. 

We  note  that  the  name  Diplodinium  caudatum  had  been  used  by 
Fiorentini  some  five  years  previously  and  that  Eberlein  was  aware 
of  this  fact,  for  he  says,  "Wenn  ich  trotzdem  die  Bezeichnung  Diplo- 
donmm  caudatum  fur  meine  Form  gewahlt  habe,  so  geschah  das  nur 
deshalb,  weil  das  Diplodinium  caudatum  Fiorentini  'identisch'  ist  mit 
dem  von  dem  gleichen  Forscher  beschriebenen  Diplodinium  rostratum 
und  desshalb  in  Fortf all  kommt. ' '  Under  the  code  of  nomenclature 
this  procedure  is  inadmissable.  In  my  paper  the  term  Diplodinium 
caudatum  refers  to  the  form  described  by  Fiorentini  under  this  name. 

To  the  work  of  Bundle  (1895)  reference  has  already  been  made. 
It  might  be  well  to  add,  however,  that  in  this  paper  Bundle  considers 
only  those  Protozoa  which  are  found  in  the  caecum  of  the  horse  and 
therefore  his  work  is  of  interest  in  this  connection  only  because  it  deals 
with  animals  more  or  less  closely  related  to  those  found  in  the  stomachs 
of  ruminants,  and  because  he  describes  from  the  caecum  of  the  horse 
a  single  ciliate,  Buetschlia  postciliata,  which  may  be  correctly  referable 
to  the  genus  Buetscklia,  described  from  the  stomach  of  ruminants. 

Giinther  (1899)  published  the  results  of  his  investigations  ^on  the 
manner  of  infection  of  ruminants  with  these  protozoans,  together  with 
a  very  complete  account  of  the  process  of  division  in  Ophryoscolex 
caudatus,  and  describes  for  the  first  time  a  new  structure  in  the  body 
of  this  animal.  This  structure  he  terms  the  "Stiitzapparat"  and 
suggests  that  it  functions  as  a  support  for  the  retractile  gullet.  To 
this  part  of  the  paper  reference  will  be  made  later. 

During  the  following  year  Giinther  (1900)  published  a  second 
paper,  dealing  this  time  with  the  finer  structure  of  some  of  the  ciliates 
both  of  the  ruminants  and  of  the  horse,  i.e.,  Ophryoscolex  caudatus, 
Entodinium  rostratum,  and  Diplodinium  [sp. ?]  from  the  sheep;  and 
Cycloposthium  bipalmatum  from  the  horse.  This  paper  is  of  especial 


1914J  Sharp:  Diplodinium  ecaudatum  49 

interest  in  that  it  contains  a  somewhat  more  complete  description  of 
the  ' '  Stiitzapparat "  in  both  Ophryoscolex  caudatus  and  Entodinium 
rostratum,  and  gives  the  first  account  of  an  observed  case  of  conjuga- 
tion in  these  ciliates,  i.e.,  in  Cysloposthium  bipalmatum,  a  ciliate  from 
the  horse.  He  says  (p.  659)  :  "  Auch  mir  ist  es  leider  nicht  gelungen, 
mehr  als  sechs  Exemplare,  die  in  Konjugation  waren,  aufzufmden. 
Desshalb  ist  es  mir  auch  unmoglich  gewesen,  die  Vorgange  wahrend 
der  Konjugation  zu  verfolgen  und  ich  kann  daher  auch  nur  wenig 
iiber  das  Faktum,  dass  ich  Konjugation  gefunden  habe,  hinausgehen. " 

Giinther  also  finds  in  Ophryoscolex  caudatus  and  Entodinium  ros- 
tratum certain  structures  which  he  describes  at  some  length  as  "myo- 
nemes. ' '  These  myonemes  he  finds  especially  at  the  bases  of  the  mem- 
branelles,  and  at  the  bases  of  the  spines.  I  can  only  say  here  that  I 
have  not  been  able  to  find  such  structures  in  the  species  Diplodinium 
ecaudatum,  but  that  contractile  fibers  are  undoubtedly  present  in  the 
esophagus  of  this  species.  This  point  will  be  fully  discussed  under  the 
description  of  the  esophagus.  . 

Some  other  European  investigators  and  writers,  Brandt  (1909), 
Liebetanz  (1910),  and  Doflein  (1911),  have,  during  the  past  few 
years,  published  on  this  group,  but  their  communications  have  dealt 
largely  with  the  physiological  relations  of  these  organisms  and  so  have 
contributed  little  to  the  solution  of  the  systematic  or  morphological 
problems  of  these  interesting  protozoans. 


PRESENT  STATUS 

Up  to  the  present  time  there  have  been  described  as  existing  in 
the  stomach  of  ruminants  some  twenty-four  species  of  protozoan 
ciliates,  classified  as  follows : 

LIST  OF  SPECIES  OF  CILIATES  DESCRIBED 
(a)   from  the  stomach  of  ruminants 

Family  A.  Ophryoscolecidae  Stein,  1858. 
Genus  I.  Ophryoscolex  Stein,  1858. 

Species     1.  Ophryoscolex  inermis  Stein,  1858. 

2.  Ophryoscolex  purkynjei  Stein,  1858. 

3.  Ophryoscolex  caudatus  Eberlein,  1895. 

Genus  II.  Diplodinium  Schuberg,  1888. 

Species     4.  Diplodinium  vortex  Fiorentini,  1889. 

5.  Diplodinium  maggii  Fiorentini,  1889. 
/  6.  Diplodinium  bursa  Fiorentini,   1889. 


50  University  of  California  Publications  in  Zoology        [VOL.  13 

7.  Diplodinium  dentatum    Schuberg,    1888,    Fiorentini    emend., 

1889. 

8.  Diplodinium  denticulatum  Fiorentini,  1889. 

9.  Diplodinium  ecaudatum  Fiorentini,  1889. 

10.  Diplodinium  caudatum  Fiorentini,  1889. 

11.  Diplodinium  rostratum  Fiorentini,  1889. 

12.  Diplodinium  cattanei  Fiorentini,  1889. 

13.  Diplodinium  caudatum  Eberlein,  1895  (non  caudatum  Fioren- 

tini). 

Genus  III.  Entodinium  Stein,  1858. 

Species  14.  Entodinium  bursa  Stein,  1858. 

15.  Entodinium  caudatum  Stein,   1858. 

16.  Entodinium  dentatum  Stein,  1858. 

17.  Entodinium  minimum  Schuberg,  1888. 

18.  Entodinium  rostratum  Fiorentini,   1889. 

Family  B.  Isotrichidae  Biitschli,  1888. 
Genus  IV.  Isotricha  Stein,  1859. 

Species  19.  Isotricha  intestinalis  Stein,  1858. 
20.  Isotricha  prostoma  Stein,  1859. 

Genus  V.  Dasytricha  Schuberg,  1888. 

Species  21.  Dasytricha  ruminantium  Schuberg,  1888. 

Genus  VI.  Buetschlia  Schuberg,  1888. 

Species  22.  Buetschilia  parva  Schuberg,  1888. 

23.  Buetschlia  neglecta  Schuberg,  1888. 

24.  Buetschlia  lanceolata  Fiorentini,  1889. 

(&)  from  the  Caecum  of  the  Horse 

Genus  I.  Cycloposthium  Bundle,  1895. 

Species     1.  Cycloposthium  bipalmatum  (Fiorentini),  1890. 

Genus  II.  Blepharyocorys  Bundle,  1895. 

Species     2.  Blepharyocorys  uncinata  (Fiorentini),  1890;  Bundle,  1895. 

3.  Blepharyocorys  valvata  (Fiorentini),  1890;  Bundle,  1895. 

4.  Blepharyocorys  unifasciculatum  (Fiorentini),  1890. 

5.  Blepharyocorys  jubata  Bundle,  1895. 

Genus  III.  Spirodinium  Fiorentini,  1890. 

Species     6.  Spirodinium  equi  Fiorentini,  1890. 

Genus  IV.  Triadinium  Fiorentini,  1890. 

Species     7.  Triadinium  caudatum  Fiorentini,   1890. 


1914]  Sharp:  Diplodinium  ecaudatum  51 

Genus  V.  Paraisotricha  Fiorentini,  1890. 

Species     8.  Paraisotricha  colpoidea  Fiorentini,  1890. 
9.  Paraisotricha  oblonga  Fiorentini,  1890. 

10.  Paraisotricha  ovalis  Fiorentini,  1890. 

11.  Paraisotricha  triangularis  Fiorentini,  1890. 

12.  Paraisotricha  ampulla  Fiorentini,  1890. 

13.  Paraisotricha  incisa  Fiorentini,  1890. 

14.  Paraisotricha  truncata  Bundle,  1895. 

Genus  VI.  Didesmis  Fiorentini,  1890. 

Species  15.  Didesmis  ovalis  Fiorentini,  1890. 

16.  Didesmis  quadrata  Fiorentini,  1890. 

Genus  VII.  Buetschlia  Schuberg,  1888. 

Species  17.  Buetschlia  postciliata  Bundle,  1895. 

Genus  VIII.  Blepharoprosthium  Bundle,  1895. 

Species  18.  Blepharoprosthium  pireum  Bundle,  1895. 

Genus  IX.  Blepharosphaera  Bundle,  1895. 

Species  19.  Blepharosphaera  intestinalis  Bundle,  1895. 

Genus  X.  Blepharocodon  Bundle,  1895. 

Species  20.  Blepharocodon  appendiculatus  Bundle,  1895. 


Diplodinium  mammosum  Railliet   (1890)   is  not  included  in  the 
list,  since  we  have  not  had  access  to  its  description. 


VALIDITY  OP  SPECIES 

Diplodinium  vortex  Fiorentini  is  undoubtedly  identical  with 
Ophryoscolex  purkynjei  Stein,  and  Diplodinium  rostratum  Fiorentini 
is  unquestionably  a  recent  division  product  of  Diplodinium  caudatum 
described  by  the  same  author.  We  have  already  referred  to  the 
validity  of  the  species  described  by  Eberlein  (1888)  as  D.  caudatum 
and  shall  refer  to  it  again  along  with  D.  vortex  Fiorentini,  and  D. 
rostratum  Fiorentini.  With  the  exception  of  Buetschlia  neglecta 
Schuberg,  Buetschlia  lanceolata  Fiorentini  and  Diplodinium  eberleini 
nom.  nov  (  =  D.  caudatum  Eberlein),  all  of  the  above  named  species 
(described  from  the  stomachs  of  ruminants)  are  present  in  the  stomachs 
of  the  sheep  and  cattle  from  the  Pacific  Coast,  i.e.,  principally  from 
California,  Nevada,  Arizona,  and  Mexico. 


52  University,  of  California  Publications  in  Zoology        [VOL.  13 

TECHNIQUE 

New  facts  are  usually  brought  to  light  by  the  discovery  of  new  or 
better  methods.  These  afford  a  solid  foundation  for  progress.  Much 
can  be  judged  of  the  scientific  value  of  results  by  a  knowledge  of  the 
methods  used  to  obtain  them.  For  this  reason  the  methods  employed 
in  procuring,  preparing,  and  studying  these  animals  are  given  in  some 
detail. 

Living  Material. — The  material  was  obtained  at  the  Oakland  Meat 
and  Packing  Company's  stockyards,  which  are  a  forty-five  minute 
car-ride  distant  from  the  laboratory.  The  first  problem  was  neces- 
sarily to  devise  a  means  of  carrying  the  stomach  fluid  which  contained 
these  parasites  from  the  slaughter-house  to  the  laboratory  without 
allowing  a  fall  in  temperature,  for  as  is  well  known,  a  loss  of  three 
or  four  degrees  Centigrade  will  cause  the  death  of  these  animals.  In 
this  connection  it  is  interesting  to  note  that  of  all  the  twenty-odd 
species  of  ciliates  described  from  the  stomachs  of  ruminants  the  Diplo- 
dinium  ecaudatum  series  is  the  most  sensitive  to  changes  of  tempera- 
ture, a  fact  not  heretofore  recorded. 

Two  points  are  kept  in  mind :  the  rapidity  of  obtaining  the 
material  after  the  animal  has  been  killed,  and  prevention  of  loss  of 
heat  from  the  container  while  on  the  way  to  the  laboratory.  We  go 
directly  to  the  killing  floor  and  as  soon  as  the  viscera  are  removed 
from  an  animal,  which  is  usually  within  three  to  five  minutes  after 
its  death,  an  assistant  removes  the  reticulum  or  ' '  honeycomb, ' '  turns  it 
inside  out  to  remove  the  partly  digested  food  material  and  then 
wrings  it  as  one  would  wring  a  wet  cloth,  into  the  operator's  hand, 
which  is  held  cupped  to  receive  the  fluid.  At  the  inner  edge  of  the 
hand,  pressed  close  to  the  flesh,  is  the  opening  of  the  container,  held 
in  such  a  manner  that  the  hand  forms  a  warm  funnel  which  Collects 
and  transfers  the  fluid  to  the  container  with  the  least  possible  loss 
of  heat.  By  using  a  "Thermos"  vacuum  bottle  contained  in  a  well 
insulated  warm  box  it  is  possible  to  convey  the  stomach  fluid  from 
the  animal  to  the  warm  oven  in  the  laboratory  with  a  fall  in  tempera- 
ture of  not  more  than  one-half  of  a  degree  Centigrade.  Schuberg 
(1888)  after  obtaining  his  material  wrapped  the  glass  container  in 
a  cloth  and  carried  it  thus  in  his  pocket  for  half  an  hour  before 
reaching  the  laboratory.  Eberlein  (1895)  collected  the  stomach  fluid 
in  test-tubes,  which  he  placed,  without  wrapping,  in  his  trouser's 
pocket,  where  he  carried  them  for  an  hour  before  reaching  the  labora- 
tory. Eberlein  notes  that  the  temperature  may  fall  as  low  as  20°  C 


1914]  Sharp:  Diplodinium  ecaudatum  53 

without  causing  death,  but,  although  this  may  be  true  for  some  of  the 
species,  it  certainly  does  not  hold  for  the  species  of  Diplodinium. 
Fiorentini  (1889)  says  nothing  of  the  manner  in  which  he  conveys 
the  stomach  fluid  to  the  laboratory  except  that  it  is  in  test-tubes,  but 
he  does  explain  how  he  keeps  the  liquid  warm  in  the  laboratory.  He 
says :  ' '  For  keeping  the  test-tubes  at  the  temperature  indicated  above 
(30  to  35°  C)  we  have  recourse  to  a  system  of  immersion  in  a  vase 
of  hot  water,  which  is  renewed  from  time  to  time,  or  still  better,  we 
keep  it  in  an  oven  at  a  constant  temperature  regulated  at  35°  C." 

Schuberg  (1888)  gives  no  detailed  description  of  his  method  of 
keeping  the  animals  alive  while  under  observation.  He  speaks  of  a 
heated  microscope  stage  ("geheizten  Objecttisch"),  but  does  not  tell 
how  he  keeps  this  stage  hot  nor  does  he  tell  how  long  he  is  able  to 
keep  alive  the  animals  under  observation.  Fiorentini  says  that  he  had 
recourse  to  the  Schultze  warm  stage,  and  notes  that  he  was  by  this 
method  enabled  to  keep  the  animals  under  observation  alive  for  a 
long  time,  "even  for  a  whole  hour  at  a  time."  Fiorentini  also  used 
another  method  which  he  explains  as  follows:  "First  I  heat  the  glass 
slide  at  a  small  lamp  until  it  is  lukewarm;  then  I  put  a  drop  of  the 
material  to  be  observed  upon  it  and  cover  it  with  a  cover  glass.  Then 
with  a  pipette  I  take  boiling  water  from  a  capsule  which  I  keep  near 
me  and  drop  this  hot  water  on  the  glass  slide  in  such  a  manner  that 
it  will  not  mix  with  the  fluid  underneath  the  coverglass.  The  hot 
water  thus  placed  upon  the  slide  allows  me  to  maintain  for  a  long 
time  the  glass  and  the  material  being  observed  at  a  sufficiently  high 
temperature  so  that  I  am  permitted  to  make  my  observations  on  the 
living  protozoans"  (translated  from  the  Italian  original).  And 
further  he  says:  "Once  the  slide  becomes  cold  one  begins  again  with 
a  new  preparation."  And  again:  "This  method  is  indispensable  dur- 
ing winter.  In  summer,  however,  it  is  sufficient  to  heat  up  the  slide 
to  get  a  preparation  which  keeps  long  enough  to  permit  one  to  make  a 
long  observation."  This  method,  Eberlein  states,  he  was  unable  to 
use;  instead  he  employed  a  glass  plate  which  he  laid  on  top  of  the 
stage  and  on  top  of  this  he  placed  his  slides.  The  glass  plate  he  heated 
by  placing  two  small  lamps  under  the  projecting  corners.  Eberlein 
says  that  he  soon  became  expert  enough  to  tell  the  proper  temperature 
of  the  glass  plate  through  the  sense  of  touch. 

Bundle  (1895)  in  his  report  on  the  ciliates  from  the  caecum  of 
the  horse  says  very  little  about  the  methods  which  he  employed  to 
the  animals  alive  under  observation  except  that  he  sometimes 


54  University  of  California  Publications  in  Zoology        [VOL.  13 

used  the  "heizbaren  Objekttisch"  of  M.  Schultze  and  sometimes  did 
not,  and  that  he  was  never  able  to  keep  the  animals  alive  for  more 
than  two  or  three  hours  at  the  most. 

Giinther  (1899  and  1900)  says  even  less  about  his  methods  than 
does  Bundle  (1895).  Both  investigators  found  that  the  alcoholic 
corrosive-sublimate  solution  gave  the  best  results  as  a  fixing  agent 
and  were  inclined  to  the  use  of  haematoxylin  as  the  most  satisfactory 
stain. 

In  my  study  of  the  living  animals  use  has  been  made  of  an  auto- 
matic constant-temperature  warm  oven  slightly  modified  from  the 
pattern  used  by  Dr.  J.  A.  Long  (1912)  in  his  study  of  the  living  eggs 
of  rats  and  mice.  The  adjustment  of  the  automatic  temperature  regu- 
lator is  such  that  the  temperature  of  the  material  on  the  slide  may  be 
kept  constant  to  within  0.5°  C.  for  an  indefinite  period  of  time.  The 
great  advantages  that  such  an  apparatus  offer  are:  first,  that  not 
only  the  material  under  actual  observation,  but  also  the  remaining 
material,  the  microscope  stage,  the  slides,  the  cover  glasses,  the  pipettes, 
etc.  may  all  be  kept  at  the  desired  constant  temperature,  and  second, 
that  the  temperature  under  wrhich  the  animals  are  being  studied,  can 
be  easily  raised  or  lowered  as  desired,  at  the  will  of  the  observer. 

When  the  fluid  to  be  studied  is  brought  from  the  slaughter  house 
to  the  laboratory,  it  is  immediately  put  into  this  warm  oven  so  that 
from  the  time  of  leaving  the  stomach  to  the  time  of  being  placed  on 
the  slide,  the  loss  in  temperature  is  not  greater  than  0.5°  C.  and  once 
on  the  slide,  the  drop  of  fluid  may  be  studied  for  from  eight  to  ten 
hours  without  any  apparent  injury  to  the  animals.  A  second  impor- 
tant use,  as  noted  above,  to  which  this  apparatus  may  be  put  and  a 
most  necessary  one  in  studying  the  movements  of  the  living  animals 
is  that  of  temperature  control.  Under  normal  conditions  the  exceeding 
great  liveliness  of  these  organisms  makes  their  accurate  study  ex- 
tremely difficult.  Various  investigators  have  recommended  the  ad- 
dition of  some  substance  such  as  a  watery  solution  of  cherry  tree  gum, 
a  three  or  four  percent  solution  of  gelatin,  Irish  moss,  quince  seeds, 
etc.,  which  would  eliminate  or  at  least  diminish  the  constant  motion  of 
these  animals.  All  of  these  methods  have  been  tried  by  me  with  more 
or  less  success,  but  with  the  inevitable  result  that  the  longevity  of  the 
organisms  has  been  decreased.  By  means  of  the  above  described  con- 
stant-temperature oven,  however,  the  operator  can  regulate  the  temper- 
ature to  such  a  degree  that  the  activity  of  the  animals  may  be  almost 
absolutely  controlled  without  any  apparent  injurious  effects.  To 


1914]  Sharp:  Diplodinium  ecaudatum  55 

avoid  the  evaporation  of  the  fluid  under  the  cover  glass,  small  dishes 
of  water  are  placed  within  the  oven,  a  plan  which  serves  to  prolong 
the  period  of  observation  to  some  degree.  Another  plan  which  I  use 
when  long  continued  observations  on  the  same  animals  are  desired  is 
to  arrange  on  the  slide  a  glass  cell  filled  with  a  drop  or  two  of  the 
stomach  fluid  and  from  which  a  fine  cotton  thread  leads  to  the  fluid 
under  observation.  The  thread  acts  as  a  siphon  and  serves  to  keep  an 
ever  fresh  supply  of  fluid  under  the  cover  glass. 

In  brief,  it  has  been  possible  by  means  of  this  apparatus  to  control 
the  rapidity  of  the  movements  of  these  animals,  through  temperature 
regulation,  and  to  keep  living  Diplodinium  under  constant  observation 
for  from  eight  to  ten  hours  at  a  time.  In  fact  the  animals  have  been 
kept  alive  for  over  forty-eight  hours  after  removal  from  the  stomach 
of  the  ox  and,  within  this  limit,  i.e.,  forty-eight  hours,  the  length  of  the 
period  during  which  an  individual  animal  may  be  kept  under  observa- 
tion, barring  accidents,  depends  entirely  upon  the  endurance  of  the 
observer. 

Fixation  and  Staining. — The  following  fixing  fluids  have  been  used 
with  good  results;  Schaudinn's  alcoholic  sublimate  solution,  Zenker's, 
Flemming's,  Worcester's,  and  Bouin's  fluids,  formalin  (4%),  and  os- 
mic  acid  (1%).  Of  these  Schaudinn's,  used  hot,  gave  uniformly  the 
best  results.  When  it  was  desired  to  follow  with  Mallory's  connective 
tissue  stain  Zenker's  fluid  was  the  best  fixing  agent.  For  the  study  of 
surface  markings  it  was  found  necessary  to  fix  in  warm  four  per  cent 
formalin  and  mount  unstained  in  styrax.  This  method  gives  excellent 
preparations. 

The  preparation  of  fixed  material  is  done  on  the  "killing  floor"  at 
the  slaughter  house.  A  table  is  arranged  as  near  to  the  spot  where 
the  cattle  are  killed  as  possible.  On  the  table  are  two  pans  of  hot  water, 
one  maintained  at  36°  C.  in  which  a  shallow  glass  dish  is  placed  and 
the  other  which  is  maintained  at  near  the  boiling  point  serves  as  a  con- 
tainer for  the  tubes  of  fixing  fluid.  The  minute  the  ox  is  opened  and 
the  stomach  removed  the  assistant  obtains  the  reticulum,  turns  it  inside 
out  in  order  to  get  rid  of  the  superfluous  food  particles,  then  quickly 
wrings  the  fluid  from  the  walls  into  the  glass  dish  in  the  water  at  36°  C. 
temperature.  Instantly  the  operator  dashes  the  boiling  Schaudinn's 
fluid  into  this  dish.  In  this  manner  the  protozoans  are  obtained  with 
cilia  extended  and  without  contractions  or  contortions  of  the  body. 
After  fixing  for  five  to  ten  minutes  the  mixture  is  shaken  up  with  twice 
its  volume  of  50  per  cent  iodine  alcohol.  This  is  repeated  until  the 


56  University  of  California  Publications  in  Zoology        [VOL.  13 

iodine  color  persists.  The  mixture  is  then  gradually  run  down  to  water 
when  the  process  of  staining  is  begun.  In  the  case  of  formalin  as  a  fix- 
ative it  is  best  to  use  it  at  about  36°  C.  as  the  boiling  formalin  has  a 
tendency  to  contract  the  endoplasm  and  ectoplasm  and  so  leave  the 
cuticle  somewhat  wrinkled.  "When  other  fixatives  are  used  the  methods 
of  handling  are  in  accordance  with  those  usually  given  for  protozoan 
fixation. 

For  in  toto  staining  the  most  satisfactory  results  are  obtained  by 
the  use  of  Heidenhain's  iron-alum  haematoxylin  solution,  as  follows: 
From  water  the  organisms  are  subjected  to  a  1  per  cent  iron-alum 
solution  for  twenty-four  to  thirty-six  hours.  Then  thoroughly  washed 
in  distilled  water,  stained  in  a  0.3  per  cent  solution  of  haematoxlyn 
(Heidenhain's)  for  twenty-four  hours  and  then  washed  in  tap  water. 
It  is  necessary  then  to  differentiate  with  a  1  per  cent  iron-alum  solution 
under  the  microscope  to  be  sure  that  differentiation  is  carried  on  to  the 
right  degree.  After  differentiation  the  animals  are  again  washed  in  tap 
water  and  distilled  water,  then  passed  up  through  the  alcohols,  xylol,  to 
cedar  oil,  and  mounted  either  in  Canada  balsam  or  styrax.  Freshly 
made  up  iron-alum  and  freshly  made  up  haematoxylin  solutions  have 
given  uniformly  better  results  than  the  ' '  ripened ' '  solutions. 

Eberlein  (1895)  suggests  the  freeing  of  the  stomach  fluid  from 
excess  food  particles  by  straining  through  a  warmed  linen  cloth.  Such 
a  method  might  result  in  the  loss  of  many  of  the  organisms.  We  have 
found  that  by  careful  manipulation  of  the  centrifuge  an  almost  pure 
culture  of  the  animals  may  be  obtained.  By  rotating  the  centrifuge  at 
the  proper  speed  the  food  particles  heavier  than  the  organisms  will  be 
thrown  down  first  while  those  lighter  than  the  organisms  will  remain 
nearer  the  top.  The  top  and  bottom  portions  may  then  be  thrown 
away.  This  process  is  of  course  carried  on  simultaneously  w'ith^hat  of 
washing  and  staining  and  if  the  top  and  bottom  portions  are  removed 
each  time  the  fluid  is  changed  the  remainder  will  soon  consist  mainly 
of  the  desired  organisms. 

Segregating. — Both  in  making  whole  mounts  and  in  sectioning  the 
animals  it  has  been  desirable  to  segregate  the  species.  This  is  accomp- 
lished by  means  of  the  following  apparatus:  A  small  hypodermic 
syringe,  fitted  with  a  glass  tube  drawn  out  to  an  almost  microscopic 
cross  section  is  clamped  to  the  microscope  stage  in  such  a  manner  that 
the  end  of  the  glass  tube  is  in  the  field  of  vision.  The  glass  tube  is  ad- 
justed and  held  in  position  by  a  screw  clamp.  Then  with  the  organisms 
in  a  cedar-oil  medium  and  the  glass  tube  and  syringe  filled  with  cedar 


Sharp:  Diplodinium  ecaudatum  57 

oil  the  operator  can  by  means  of  the  mechanical  stage  bring  a  desired 
animal  up  to  the  end  of  the  glass  tube  into  which  it  may  be  sucked  by 
a  slight  twist  of  the  piston  of  the  syringe.  In  this  way  the  desired 
animals  may  be  sorted  out  free  from  dirt  and  other  animals  to  the 
number  of  from  two  hundred  and  fifty  to  five  hundred  in  a  single  tube. 
By  substituting  a  fresh  slide  on  which  is  placed  a  drop  of  Canada  bal- 
sam or  styrax  the  animals  may  be  easily  ejected  from  the  glass  tube  into 
the  mounting  substance. 

By  mounting  these  animals  between  two  thin  cover  glasses  and  then 
fixing  the  cover  glasses  in  a  brass  frame  which  is  constructed  to  hold 
them  as  a  window  frame  holds  the  pane  of  glass,  the  same  individual 
animal  may  be  viewed  from  either  side.  This  simple  bit  of  apparatus 
has  proved  itself  exceedingly  useful  in  determining  many  otherwise 
difficult  problems  and  is  therefore  recommended  for  consideration  to 
those  making  similar  investigations. 

Sectioning. — When  it  is  desired  to  section  the  animals  they  are 
segregated  by  the  above  method  and  ejected  from  the  glass  tube  into 
small  gelatin  capsules  (such  as  may  be  obtained  at  any  pharmacy) 
which  have  been  partially  filled  with  imbedding  paraffin.  The  capsule 
is  placed  in  a  warm  oven  at  the  proper  temperature,  the  paraffin  melts, 
the  cedar  oil  diffuses  through  the  paraffin,  and  the  animals  sink  to  the 
bottom.  By  soaking  in  water  for  a  few  minutes  the  gelatin  capsule 
may  be  easily  slipped  off  and  by  cutting  off  the  bottom  end  each  time 
and  running  this  through  another  paraffin  filled  capsule  for  at  least 
four  times,  a  paraffin  infiltration  of  the  organisms,  sufficient  to  allow 
of  sectioning,  is  obtained.  When  the  infiltration  is  complete  the  lower 
four  or  five  drops  of  paraffin  containing  the  animals  are  withdrawn  by 
means  of  a  warm  capillary  pipette  and  run  into  a  mould  which  has 
been  prepared  in  the  following  manner.  A  small  brass  rod  is  filed  to  a 
square  cross  section  and  fitted  in  an  imbedding  plate  so  that  when  the 
paraffin  is  poured  around  it,  it  will  act  as  a  core.  When  the  paraffin 
hardens  and  the  core  is  removed  a  perfect  mould  is  obtained.  Paraffin 
of  a  much  higher  melting  point  should  be  used  for  the  moulds  than 
that  which  is  used  for  imbedding,  and  if  the  paraffin  of  the  mould  be 
slightly  tinted  by  Sudan  III.,  the  liability  of  cutting  into  the  imbed- 
ding paraffin  is  eliminated.  When  the  imbedding  paraffin  containing 
the  animals  is  run  into  the  mould  care  should  be  taken  that  the  animals 
are  well  distributed  through  the  paraffin  and  that  the  paraffin  is  just 
above  the  melting  point.  It  then  hardens  before  the  animals  have  time 
to  sink  to  the  bottom,  and  so  results  in  their  remaining  well  distributed 


58  University  of  California  Publications  in  Zoology        [VOL.  13 

throughout  the  block.  By  this  method  very  little  practice  is  necessary 
in  order  to  obtain  a  perfect  imbedding.  The  advantages  of  such  a 
method  are:  (1)  the  certainty  that  only  the  desired  animals  are 
present;  (2)  the  ease  of  handling;  (3)  perfect  infiltration;  (4)  even 
distribution;  (5)  the  production  of  a  block  with  a  smaller,  truer, 
square  cross  section  than  it  is  possible  to  obtain  by  trimming  by  hand, 
a  point  which  is  very  important  when  good  ribbons  are  necessary.  In 
some  respects  it  is  advantageous  to  stain  in  toto  before  sectioning,  but 
the  best  results  are  obtained  by  staining  the  sections. 

Section  Staining. — For  section  staining  Mallory's  connective  tissue 
stain  not  only  gives  excellent  results — giving  four  distinct  colors— but 
also  reveals  a  very  surprising  structural  differentiation  which  to  my 
knowledge  has  not  before  been  described. 

The  slides  should  be  run  from  xylol  very  gradually  down  to  distilled 
water. 

Sec. 

Fuchsin  S.,  aqueous  solution   (%%)    45 

Distilled  water 5 

Phospho-molybdic  acid  (1%)  - 60 

Fresh  distilled  water 5 

Anilin  blue,  orange  G.  and  oxalic  acid  60 

Distilled  water  10 

95%  alcohol  1 

100%  alcohol  1 

Carbo-xylol    1 

Xylol, 
Mount. 

If  the  above  method  is  carefully  followed  preparations  may  be  ob- 
tained in  which  the  cilia  show  clear  i.e.,  transparent,  the  ectoplasm  a 
blue-red,  the  entoplasm  pink,  the  macronucleus  orange-brown  and  the 
micronucleus  and  some  fibers  which  will  be  described  later^how  bright 
red  by  transmitted  light. 

Heidenhain's  iron-alum  haematoxylin  made  up  as  described  above 
for  in  toto  staining  also  gives  very  perfect  preparations. 

Genus  Diplodinium  Schuberg  1888 

Schuberg  (1888),  p.  404. 

Fiorentini  (1889),  pp.  11-17,  pi.  1,  figs.  1-4;  pi.  2,  figs.  1-5;  pi.  3,  figs.  1-5. 

Eberlein  (1895),  pp.  251-264,  pi.  17,  figs.  8-17;  pi.  18,  figs.  18-20. 

The  genus  Diplodinium  was  separated  from  the  genus  Entodinium 
of  Stein  (1858)  by  Schuberg  (1888).  Although  Schuberg  set  up  this 
new  genus  he  gives  a  very  unsatisfactory  characterization  of  it.  He 


1914] 


Sharp:  Diplodinium  ecaudatum 


59 


says  (p.  404)  :  "Stein  hat  unter  dem  Gattungsnamen  Entodinium  drei 
Infusorienformen  vereinigt,  die  ich  mit  ziemlicher  Sicherheit  auch 
angetroffen  habe.  Ein  genaueres  Studium  ergab  jedoch,  dass  eine 
dieser  Arten,  Entodinium  dentatum,  wie  Ophryoscolex,  eine  zweite 
Wimperzone  besitzt,  und  dass  auch  Thieren,  die  mit  Entod.  bursa 
Stein  eine  gewisse  Aehnlichkeit  haben,  und  die  Stein  weniger  iiber- 
sehen,  als  mit  dieser  letztern  zusammengeworfen  zu  haben  scheint, 
eine  solche  zukommt.  Ich  trenne  die  Formen,  die  Entodinium  im  all- 
gemeinen  gleichen,  jedoch  durch  eine  zweite  Wimperzone  ausgezeichnet 
sind,  unter  dem  Namen  Diplodinium  ab ;  dieselben  sind  iibrigens  auch 
noch  durch  andere  Merkmale  als  naher  zusammen  gehorig  character- 
isirt,  worauf  aber  hier  noch  nicht  naher  eingegangen  werden  soil. ' ' 

That  this  definition  is  insufficient  will  be  seen  when  we  consider 
the  genus  Opliryoscolex,  the  members  of  which  also  resemble  Ento- 
dinium and  possess  two  membranelle  zones,  but  which  are  not  members 
of  the  genus  Diplodinium.  Some  of  the  more  apparent  differences 
between  the  three  genera  are  given  in  the  following  table : 


*Mean   dimensions 

of   the  body 

in  mm. 

Examples  Length  Width 

O.  inermis  Stein  0.180  0.085 

O.  caudatus  Eberlein  0.180  0.085 

O.  purkynjei  Stein  0.175  0.085 

D.  bursa  Fiorentini  0.120  0.065 

D.  caudatum  (=  eberleini 

nom.  nov.)  0.105  0.065 

D.  eeaudatum  Fiorentini  0.055  0.025 

E.  bursa  Stein                              0.090  0.050 
E.  caudatum  Stein                      0.080  0.040 
E.  dentatum  Stein                       0.075  0.040 

*  Eberlein  (1895),  p.  283. 


Dorsal 

membranelle  zone  Vacuoles 

Incomplete  spiral  en- 
circling  %    of  the  5-6 
entire  body. 

Transverse,  encircling 

less  than  %  of  the  2-4 

entire  body. 


Absent. 


Schuberg  (1888),  so  far  as  I  am  able  to  determine,  has  never  given 
any  descriptions  of  individual  species  of  this  genus,  but,  as  quoted 
above,  merely  cites  Entodinium  dentatum  Stein  as  belonging  to  this 
new  genus,  along  with  another  form  which  resembles  Entodinium 
bursa  and  which,  in  my  opinion,  may  probably  be  Diplodinium  bursa 
Fiorentini  (1889).  Stein  (1858)  describes  Entodinium  bursa  (type 
specie,?  of  the  genus  by  location),  E.  dentatum,  and  E.  caudatum, 


60  University  of  California  Publications  in  Zoology        [VOL.  13 

species  which  still  hold  good  in  that  genus,  but  gives  no  figures,  and 
Schuberg  (1888)  gives  neither  a  description  nor  a  figure  of  the  species 
of  Diplodinium  to  which  he  attached  the  name  " dentatum."  Stein 
(1858)  explicitly  states  that  "der  Wimpergiirtel  der  Riickseite  fehlt 
jedoch"  in  Entodinium.  Schuberg  (1888)  founds  Diplodinium  upon 
a  ciliate  with  a  dorsal  membranelle  zone.  There  is,  therefore,  no  ques- 
tion but  that  he  had  before  him  as  the  species  upon  which  he  founded 
his  new  genus  a  specimen  different  from  Entodinium  dentatum  Stein 
and  correctly  referable  to  his  new  genus  Diplodinium  because  it  had 
a  dorsal  membranelle  zone.  Therefore  E.  dentatum  Stein  and  Diplo- 
dinium dentatum  Schuberg  are  two  different  species  in  different 
genera  and  both  names  are  valid,  although  Schuberg 's  opinion  as  to 
the  identity  of  the  two  species  implied  in  his  statement  above  quoted 
and  in  his  use  of  Stein's  specific  name  is  in  error.  This  decision  is  in 
accord  with  the  usage  of  Eberlein  (1895).  The  question  as  to  the 
type  species  of  Diplodinium  is  a  very  complicated  one.  It  seems  wisest, 
however,  in  view  of  the  subsequent  history  of  the  case,  to  accept  Schu- 
berg's  D.  dentatum  as  later  described  and  figured  by  Fiorentini  (1889) 
as  the  type  species.  Fiorentini  (1889)  utilized  Schuberg 's  name  for 
this  inadequately  defined  and  still  less  adequately  established  genus 
and  assigns  to  it  nine  species,  which  he  figures  and  describes  as  Diplo- 
dinium vortex,  D.  maggii,  D.  bursa,  D.  dentatum  Schuberg,  D.  denti- 
culatum,  D.  ecaudatum,  D.  caudatum,  D.  rostratum  and  D.  cattanei. 
To  these  Eberlein  (1895)  added  a  new  species  which  he  named  Diplo- 
dinium caudatum,  thus  making  ten  species  described  for  this  genus. 

Diplodinium  vortex,  however,  as  is  pointed  out  by  Eberlein  (1895), 
is  not  a  member  of  the  genus  Diplodinium  at  all,  but  is  identical  with 
Ophryoscolex  purkynjei  Stein  (1858).  There  is  some  question  also 
regarding  the  specific  standing  of  D.  maggii  Fiorentini.^  Eberlein 
accepts  it  with  some  hesitation.  He  says  of  it:  "Es  ist  ziemlicb 
gross,  und  diese  Eigenschaft,  besonders  aber  die  unverhaltnismassige 
Breite  unterscheidet  es  von  Diplodinium  bursa.  Es  bleibt  zweifelhaft, 
ob  es  auf  Grund  dieser  einen  Eigenschaft  berichtigt  ist,  Diplodinium 
maggii  als  selbstandige  Art  zu  betrachten,  oder  ob  es  Diplodinium 
bursa  zuzurechen  ist. ' '  I  have  found  it  to  be,  however,  in  my  material, 
a  perfectly  well-defined  species. 

We  also  call  attention  to  the  fact  that  D.  rostratum  Fiorentini  is 
merely  an  individual  D.  caudatum,  described  shortly  after  transverse 
division,  and  so  drops  into  the  synonymy  of  the  latter.  This  leaves 
to  the  credit  of  Fiorentini  seven  species  of  the  genus  Diplodinium,  all 


19141  Sharp:  Diplodinium  ecaudatum  61 

of  which  are  present  in  my  material  and  have  been  identified  by  me. 
We  have  already  noted  the  inadmissible  procedure  of  Eberlein  (1895) 
in  assigning  the  species  D.  caudatum  Fiorentini  to  D.  rostratum  Fioren- 
tini  and  then  appropriating  the  name  D.  caudatum  for  a  species  of  his 
own.  This  species,  D.  caudatum  Eberlein,  I  have  not  yet  identified, 
but  for  purposes  of  conformity  to  the  code  of  nomenclature  I  propose 
for  it  the  name  Diplodinium  eberleini.  Granted  that  this  species, 
Diplodinium  eberleini,  is  valid,  then  the  genus  Diplodinium  contains 
up  to  the  time  of  this  paper  eight  valid  species  or  forms,  viz. :  D. 
maggii  Fiorentini,  D.  bursa  Fiorentini,  D.  dentatum  Schuberg  (Fio- 
rentini emend.),  D.  denticulatum  Fiorentini,  D.  ecaudatum  Fiorentini, 
D.  caudatum  Fiorentini,  D.  cattanei  Fiorentini,  and  D.  eberleini  nom. 
nov. 

My  observations  lead  to  the  conclusion  that  D.  denticulatum  is 
merely  a  variant  form  of  D.  dentatum  and  that  D.  caudatum  and 
D.  cattanei  are  only  forms  of  D.  ecaudatum.  In  fact  it  is  one  of  the 
purposes  of  the  present  paper  to  show  that  D.  ecaudatum  Fiorentini, 
D.  caudatum  Fiorentini  and  D.  cattanei  Fiorentini  are  forms  of  the 
species  whose  lawful  name  is  Diplodinium  ecaudatum  Fiorentini.  To 
this  species  (D.  ecaudatum  Fiorentini)  I  add  three  new  forms  to  which 
I  have  given  the  descriptive  names  D.  ecaudatum  forma  bicaudatum, 
D.  e.  forma  tricaudatum,  and  D.  e.  forma  quadricaudatum.  Diplo- 
dinium caudatum  therefore  becomes  D.  e.  forma  caudatum,  and  D. 
cattanei,  in  accordance  with  facts  wrhich  will  be  given  later,  becomes 
D.  e.  forma  cattanei.  These  three  forms  ( D.  e.  forma  ecaudatum,  D.  e. 
forma  caudatum,  and  D.  e.  forma  cattanei),  together  with  my  three 
new  forms,  constitute  a  complete  series  ranging  from  D.  e.  forma  ecau- 
datum without  posterior  spines  up  to  D.  e.  forma  cattanei  with  five 
spines.  Allowing  for  normal  variations  in  details  of  structure,  the 
morphology  of  these  six  forms  is  identical,  with  the  exception  only  of 
the  presence  and  number  of  these  spines  and  the  necessary  differences 
in  the  form  of  the  posterior  end  wrhich  their  presence  or  absence 
occasions.  From  all  the  other  species  of  Diplodinium,  however,  they 
differ  considerably.  These  facts  seem  to  warrant  the  placing  of  these 
six  forms  in  a  single  species,  which  in  accordance  with  the  rules  of 
nomenclature  must  be  designated  as  Diplodinium  ecaudatum,  although 
many  of  the  individuals  have  from  one  to  five  ' '  caudal ' '  appendages. 


62  University  of  California  Publications  in  Zoology        [VOL-  13 

1.  Diplodinium  ecaudatum   forma  ecaudatum    Fiorentini 

PI.  3,  figs.  1,  2;  pi.  4,  figs.  3-5;  pi.  6,  figs.  11-19;  pi.  7,  figs.  20-33. 
Diplodinium  ecaudatum  Fiorentini  (1889),  pp.  15-16,  pi.  3,  fig.  1. 
Diplodinium  ecaudatum,  Eberlein  (1895),  pp.  263-267,  pi.  18,  fig.  19. 

Of  all  the  various  forms  of  Diplodinium  ecaudatum  found  in  the 
stomachs  of  western  cattle  the  most  numerous  and  the  simplest  morph- 
ologically is  the  forma  ecaudatum.  This  form  is  almost  universally 
present  and  when  once  identified  may  be  easily  distinguished  from 
all  other  forms.  It  is  the  basis  of  the  following  full  description,  which 
is  applicable  to  the  other  forms  except  only  in  the  region  of  the  pos- 
terior spines. 

The  form  of  the  body  is  constant  (pi.  3,  figs.  1,  2),  somewhat  over 
twice  as  long  as  wide,  consistently  circular  in  cross-section,  obliquely 
rounded  off  at  the  anterior  and  pointedly  at  the  posterior  end  of  the 
body.  In  general  the  body  somewhat  resembles  a  rather  short,  plump 
banana,  the  dorsal  side  being  convex  and  the  ventral  slightly  concave. 
This  resemblance  would  be  still  greater  if  the  stem  end  of  the  banana, 
which  corresponds  in  position  to  the  oral  opening,  be  held  stationary 
while  the  posterior  extremity  is  twisted  slightly  to  the  right  of  the 
median  ventral  line.  The  organs  of  food-taking  and  locomotion  are 
situated  in  the  anterior  one-fourth  of  the  body,  which  part  is  more 
or  less  flexible  and  decidedly  contractile.  The  remaining  three-fourths 
of  the  body  is  rigid,  friable  and  comparatively  smooth,  i.e.,  free  from 
appendages  of  any  description.  The  anus  is  situated  at  the  posterior 
extremity  of  the  body  close  to  the  ventral  side. 

The  structure  of  the  body  is  very  complicated.  It  shows  plainly 
(fig.  B;  pi.  4,  figs.  3-5,  and  pis.  6,  7,  figs.  11-33)  a  cuticle  (cut.}, 
an  ectoplasm  (ect.},  and  an  entoplasm  (ent.},  with  a  boundary  layer 
(bd.  I.)  which  separates  the  ectoplasm  from  the  entoplasm.  ^ 

The  separate  structures  which  are  ectoplasmic  in  their  origin  and 
location  are:  three  skeletal  areas  with  underlying  skeletal  structures 
(I.  sk.  a.,  v.  sk.  a.,  and  r.  sk.  a.),  a  macronucleus  (mac.},  a  micronucleus 
(mic.},  a  motor  mass  (TO.  m.},  motor  fibers  (d.  m.  str.},  a  circum- 
oesophageal  ring  (circ.  oes.  ring},  oesophageal  retractor  strands  (oes. 
retr.  str.},  a  dorsal  row  of  membranelles  (d.  m.},  an  adoral  row  of 
membranelles  (ador.  m.},  operculum  (op.},  oral  cilia  (or.  oil.}, 
mouth  (or.},  oesophagus  (oes.},  caecum  (caec.},  rectum  (rect.},  anus 
(an.},  and  contractile  vacuoles  (ant.  c.  v.  and  post.  c.  v.}.  The  ento- 
plasm (ent.}  is  structureless  with  the  exception  of  the  contained  food 
vacuoles  (fd.  vac.}  and  food  particles. 


Sharp:  Diplodinium  ecaudatum  63 

THE  ECTOPLASMIC  STRUCTURES 

Cuticle. — The  thin  but  resistant  cuticle  covers  the  entire  body 
and  is  so  highly  specialized  over  certain  regions  (sk.  a.,  pi.  3,  figs.  1-2) 
as  to  demand  special  description.  There  are  three  such  regions  over 
which  the  cuticle  shows  a  well-defined  differentiation.  (1)  Over  the 
dorsal  (D.)  and  left  surfaces  of  the  body  the  cuticle  is  comparatively 
smooth,  giving  only  faint  evidence  of  longitudinal  striations  and  in 
places  traces  of  mottling.  (2)  The  cuticle  over  the  ventral  surface 
(V.),  extending  from  the  middle  to  the  posterior  extremity  of  the 
body  and  over  the  spines  when  present,  is  faintly  but  distinctly  mot- 
tled. This  mottling  is  caused  by  minute  diamond-shaped  depressions 
in  the  cuticle  which  at  certain  levels  do  not  transmit  the  light  as 
readily  as  the  non-depressed  portions,  and  hence  appear  as  little 
shadows.  (3)  The  third  region,  embracing  the  anterior  one-half  of 
the  left  ventral  and  ventral  surfaces  and  the  anterior  two-thirds  of 
the  right  surface,  is  divided  into  three  well-defined  areas  by  under- 
lying ectoplasmic  structures  which  appear  to  be  skeletal  in  function. 
Eberlein  (1895,  p.  240)  says  in  his  description  of  Ophryoscolex  iner- 
mis:  "Die  Ran  der  der  Bauchflache  sind  beiderseits  durch  einen 
Streifen  starker  granulierten  Plasmas  begrenzt  (fig.  1),"  but  makes 
no  mention  of  a  definite  underlying  structure.  Erlanger  (1890,  p. 
654)  observed,  in  Chlamydodon  mnemosyne  Stein,  a  strange  "Band" 
situated  between  the  dorsal  and  ventral  surfaces,  surrounding  the 
entire  body  with  the  exception  of  a  small  interruption  at  the  posterior 
end,  and  which  was  clearly  marked  off  by  cross  striations.  And 
Levander  (1894,  pp.  66-67)  in  his  description  of  Plagiopyla  nasuta 
Stein  calls  attention  to  a  previously  unnoticed  cross-striped  band  of 
differentiated  ectoplasm  situated  on  the  right  side  of  the  body  near 
to  and  parallel  with  the  ventral  border. 

The  above-mentioned  cases  of  ectoplasmic  differentiation  might 
be  conceived  as  being  similar  to  the  peculiar  ectoplasmic  differentia- 
tions which  are  described  in  this  paper  as  skeletal  areas.  In  none 
of  the  above  cases,  however,  is  anything  said  about  a  specially  differ- 
entiated underlying  ectoplasmic  structure. 

To  GHinther  (1899)  belongs  the  credit  for  being  the  first  to  note 
and  describe  this  underlying  ectoplasmic  structure.  He  says  (p. 
553)  :  "An  jedem  Ophryoscolex  caudatus,  schon  bei  der  Untersuch- 
ung  des  lebenden  Thiers,  besser  natiirlich  an  Konservirten  gefarbten 
Thieren  und  Schnitten  derselben,  habe  ich  ein  Organ  (cf.  figs.  1,  2,  5, 
6,  7  st\)  gefunden,  im  Ectoplasma  liegend,  iiber  das  bis  jetzt  noch  kein 


64  University  of  California  Publications  in  Zoology        [VOL.  13 

Autor  in  der  iiber  die  Infusorien  des  Wiederkauermagens  handelden, 

mir  zuganglichen  Litteratur  etwas  bemerkt  hat Nach  meinen 

Beobachtungen  stellt  dasselbe  einen  Stiitzapparat  fiir  den  Schlund 
dar. ' '  And  again  in  his  paper  of  1900  he  adds  that  he  has  also  found  a 
corresponding  structure  in  Entodinium  rostratum  Fiorentini.  But 
in  neither  of  these  papers  does  Giinther  (1899  and  1900)  make  men- 
tion of  a  surface  differentiation  of  the  cuticle  corresponding  to  the 
underlying  ectoplasmic  structures  ("Stiitzapparat").  As  a  matter  of 
fact  these  peculiar  structures  (sk.  lam.,  pi.  4,  figs.  3,  4,  5)  with  their 
overlying  areas  (1.  sk.  a.,  v.  sk.  a.,  and  r.  sk.  a.,  pi.  3,  figs.  1,  2)  which 
I  have  found  in  Diplodinium,  and  which  appear  to  be  skeletal  in 
function,  and  are  so  designated  in  this  paper,  are  so  different  from 
anything  heretofore  mentioned  that  a  rather  complete  description  is 
deemed  in  order. 

Skeletal  Areas. — The  skeletal,  structures  proper  will  be  described 
in  connection  with  the  ectoplasm.  The  corresponding  areas  of  cuticle, 
which  will  be  designated  as  a  left  skeletal  area,  a  ventral  skeletal  area, 
and  a  right  skeletal  area,  show  well-defined  boundaries. 

The  left  skeletal  area  (1.  sk.  a.,  pi.  3,  fig.  2),  the  smallest  of  the 
three,  is  triangular  in  shape  with  its  base  anteriorly  marked  off  by 
a  line  drawn  horizontally  from  the  left  extremity  of  the  dorsal  mem- 
branelle  zone  (d.  m.  z.,  pi.  3,  fig.  2)  to  meet  the  adoral  membranelle 
zone  (ador.  m.  z.,  pi.  3,  fig.  2)  near  its  left  extremity,  and  its  apex 
extending  obliquely  posteriorly  and  toward  the  right  to  end  on  the 
ventral  side  about  midway  between  mouth  and  anus.  The  ventral 
skeletal  area  (v.  sk.  a.,  pi.  3,  figs.  1,  2),  the  largest  of  the  three  areas, 
is  rectangular  in  shape  and  extends  from  the  base  of  the  outer  adoral 
lip  (pi.  3,  figs.  1.  2)  somewhat  obliquely  towards  the  posterior  end  and 
slightly  towards  the  right  to  blend  with  the  right  skeletal  a^ea  just 
anterior  to  the  middle  of  the  body.  The  right  skeletal  area  (r.  sk.  a., 
pi.  3,  fig.  1),  intermediate  in  size,  also  somewhat  triangular  in  shape, 
with  base  marked  off  by  a  line  extending  from  the  right  extremity 
of  the  dorsal  membranelle  zone  to  the  dorsal  side  of  the  base  of  the 
outer  adoral  lip,  and  apex  extending  posteriorly,  blends  with  the 
ventral  skeletal  area  just  anterior  to  the  middle  of  the  body.  The 
two  areas  then  extend  posteriorly  to  terminate  rather  indistinctly 
at  about  the  last  fourth  of  the  body.  The  relation  of  skeletal  areas  to 
underlying  skeletal  structures  may  be  seen  best  by  a  consideration  of 
plate  4,  figure  4,  and  plate  7,  figures  23-29.  The  cuticle  over  these 
areas  is  more  transparent  than  elsewhere  and  through  it  may  be  seen 


1914]  Sharp:  Diplodinium  ecaudatum  65 

the  underlying  skeletal  structures.  It  is  thrown  into  fine  longitudinal 
ridges  which  mark  off  the  little  diamond-shaped  depressions,  which, 
as  already  pointed  out,  give  the  mottled  appearance  so  characteristic 
of  these  areas. 

At  the  anus  (an.,  pi.  4,  fig.  3)  the  cuticle  is  continuous  with  the 
rectal  sheath  and  at  the  mouth  with  the  lining  of  the  oral  cavity  and 
oesophagus  (or.,  pi.  4,  fig.  3).  At  the  dorsal  and  adoral  zones  of  cilia 
it  dips  down  into  the  furrows  (pi.  4,  fig.  3)  and  covers  the  lips,  disks, 
and  operculum.  Over  the  lips  and  operculum  it  is  much  thickened, 
but  in  the  furrows  and  over  the  disks  it  is  thin. 

Ectoplasm. — The  ectoplasm  (ect.,  pi.  4,  figs.  3-5),  which  is  entirely 
covered  by  the  cuticle  and  separated  from  the  entoplasm  by  the  very 
distinct  boundary  layer  (bd.  I.},  is  not  a  homogeneous  substance,  but 
shows  a  rather  definite  alveolar  stroma  highly  modified  in  certain 
regions,  as  noted  above,  to  form  skeletal  structures.  This  layer  of 
ectoplasm  varies  much  in  thickness  in  different  parts  of  the  body, 
being  very  much  the  thickest  in  the  anterior  region  (ect.,  pi.  4,  figs. 
3,  4;  pi.  6,  figs.  14-16;  pi.  7,  figs.  23-27),  and  thinnest  over  the  left 
side  (ect.,  pi.  4,  fig.  5.)  In  the  anterior  and  posterior  ends  of  the 
body  the  thick  ectoplasm  fills  in  the  inequalities  of  the  outer  form  of 
the  body,  so  that  the  enclosed  entoplasm  is  smoothly  rounded  off,  and 
in  these  regions  does  not  conform  to  the  general  contour  of  the  body. 
Here  also  the  reticular  structure  is  coarser,  the  meshes  appear  larger, 
and  may  therefore  be  more  easily  studied.  That  part  which  lies  close 
to  the  cuticle  loses  its  irregular  arrangement  and  forms  a  rather  well- 
defined  alveolar  layer  in  which,  in  cross-sections,  the  alveoli  appear 
irregularly  quadrilateral.  A  similar  layer  in  which  the  individual 
alveoli  may  be  even  more  easily  distinguished  lies  next  to  the  boundary 
layer  (bd.  I.,  pi.  4,  fig.  4).  The  ectoplasm  also  encloses  the  first  part 
of  the  oesophagus  (oes.,  pi.  4,  figs.  3-4),  the  rectum  (rect.,  pi.  4, 
fig.  3),  macronucleus  and  micronucleus  (mac.  and  mic.,  pi.  4,  figs. 
3,  5),  and  the  two  contractile  vacuoles  (ant.  c.  v.  and  post.  c.  v.,  pi.  4, 
fig.  3).  It  is  noteworthy  that  in  the  immediate  vicinity  of  the  con- 
tractile vacuoles  the  alveolar  structure  is  again  modified,  in  that  the 
meshes  are  larger  and  the  interalveolar  walls  are  finer  in  structure  and 
stain  less  heavily  than  the  remaining  ectoplasm  (c.  v.  r.,  pi.  4,  fig.  3; 
see  also  microphotograph,  pi.  7,  fig.  27).  No  streaming  whatever  of 
the  ectoplasm  has  been  observed. 

Skeletal  Structures. — The  skeletal  structures  noted  in  the  descrip- 
tion >f  the  cuticle  are  undoubtedly  of  ectoplasmic  origin,  but  if,  as 


66  University  of  California  Publications  in  Zoology        [VOL.  13 

Eberlein  (1895,  p.  243)  points  out,  the  brittleness  of  the  cuticle  is 
due  to  the  presence  of  silicic  acid,  there  is  probably  silicic  acid  present 
in  this  skeletal  structure,  for  of  all  the  structures  of  the  body  this  is 
at  once  the  most  rigid  and  the  most  brittle.  The  appearance  in 
surface  view  of  these  areas  has  already  been  described.  In  cross- 
sections  each  of  these  regions  is  somewhat  elliptical,  transversely 
crossed  by  thin,  paired  laminae  (sk.  lam.,  pi.  4,  figs.  3-5;  see  also 
microphotographs,  pi.  7,  figs.  23-29),  extending  from  the  cuticle  per- 
pendicularly inward  to  the  inner  wall  of  the  elliptical  space.  At  their 
inner  and  outer  attachments  the  members  of  each  pair  of  laminae  are 
in  close  juxtaposition,  but  in  the  middle  they  spread  apart,  leaving  a 
minute  elliptical  interval  between  the  two  laminae.  The  appearance 
in  cross-sections  is  as  though  the  inner  wall  of  the  elliptical  space  were 
held  away  from  the  outer  wall  by  a  row  of  spindles,  the  longest  of 
which  is  situated  in  the  middle.  These  pairs  of  laminae  extend  ob- 
liquely posteriorly,  corresponding  with,  and,  as  a  matter  of  fact, 
causing  the  longitudinal  ridges  in  the  cuticle  over  the  above-described 
skeletal  structures.  The  central  skeletal  laminae  of  the  left  skeletal 
structure,  passing  from  anterior  to  posterior,  at  first  grow  longer  and 
longer,  pushing  the  central  portion  of  the  inner  wall  of  the  ellipse 
farther  away  from  the  outer  wall  until  what  was  an  elliptical  area 
becomes  triangular  in  outline  with  apex  pointed  toward  the  longi- 
tudinal axis  of  the  body  (pi.  7,  figs.  23-28).  Just  above  the  middle 
of  the  body,  however,  the  reverse  takes  place,  the  central  laminae 
grow  gradually  shorter  and  allow  the  inner  wall  of  the  ellipse  to 
approach  the  outer  wall  until  at  about  the  middle  of  the  body  the  left 
skeletal  structure  joins  the  ventral  skeletal  structure,  which  at  about 
the  same  level  joins  the  right  skeletal  structure,  i.e.,  the  three  skeletal 
structures  merge  into  one.  The  laminae  of  the  left  skeletal  structure 
(pi.  7,  fig.  29)  are  at  this  level  much  longer  than  those  of  the  ventral 
and  right  skeletal  structures,  but  they  now  become  rapidly  shorter, 
soon  disappear  altogether,  and  this  elliptical  space  with  its  contained 
skeletal  structure  is  entirely  obliterated  (pi.  7,  figs.  30-32).  The 
skeletal  elliptical  spaces  of  the  ventral  and  right  sides  fuse  immedi- 
ately anterior  to  the  middle  of  the  body  (pi.  7,  fig.  28).  The  laminae 
soon  become  shorter,  are  placed  more  closely  together,  and  appear  in 
cross-sections  as  a  row  of  little  pillars  which  support  the  oesophagus 
and  keep  it  away  from  the  right  wall  of  the  body  (v.  sk.  lam.  and 
r.  sk.  lam.,  pi.  4,  fig.  5,  and  pi.  7,  fig.  29).  These  laminae  also 
grow  narrower  and  narrower  up  to  the  point  immediately  anterior 


1914]  Sharp:  Diplodinium  ecaudatum  67 

to  the  last  one-fourth  of  the  body,  where  they,  too,  disappear.  It  is 
to  be  noted  that  all  traces  of  oesophageal  structure  are  lost  at  about 
the  same  level  as  that  at  which  the  united  ventral  and  right  skeletal 
structures  disappear. 

That  the  above  described  structure  functions  as  a  true  skeletal 
(supporting)  structure,  not  only  for  the  retractile  oseophagus  but 
also  for  the  entire  body,  seems  altogether  certain.  In  the  consideration 
of  the  oesophagus  and  the  motile  anterior  end  of  the  body  additional 
evidence  will  be  given  for  this  conclusion. 

Giinther  (1899,  p.  553)  describes  a  homologous  structure  for 
Ophryoscolex  caudatus,  which  is  composed  of  two  parts,  at  first  sep- 
arated one  from  the  other,  partly  embracing  the  gullet  and  then 
approaching  each  other  quite  closely,  finally  to  separate  again  and 
end  singly,  deep  down  in  the  lower  part  of  the  animal.  In  the  case 
of  Ophryoscolex  caudatus  this  structure  is  described  as  being  com- 
posed of  two  layers,  a  fine,  thin,  structureless  membrane  and  an  en- 
closed substance  which  shows  a  definite  structure,  "mit  grofien,  meist 
zur  Langsachse  des  Stiitzsapparates  senkrecht  gerichteten  Waben." 
And  in  his  description  of  a  similar  structure  in  Entodinium  rostratum 
Giinther  (1900,  p.  644)  says:  "Dicht  unter  der  Pellicula  im  oberen 
Theil  des  Thiers  beginnend,  rechts  vom  Kern  liegend,  zieht  sich  das- 
selbe  (Fig.  13  st)  in  ziemlich  breiter  Ausdehnung  bis  zur  Mitte,  wo  es 
sich  in  3  Theile  theilt  (Fig.  14  st),  von  denen  jeder  einzeln  tief  unten 
im  Thier  endet,  stets  der  Pellicula  anliegend. "  Of  the  finer  structure 
of  the  apparatus  in  this  case  nothing  is  said. 

It  is  hardly  necessary  to  point  out  that  this  description  of  the 
"Stiitzapparat"  in  Ophryoscolex  caudatus  has  little  in  common  with 
the  above  description  of  the  skeletal  structure  in  Diplodinium  ecau- 
datum except  in  general  indications  of  homology. 

Boundary  layer. — Separating  the  ectoplasm  from  the  entoplasm  is 
a  constant  and  well-defined  boundary  layer  (bd.  I.,  pi.  4,  figs.  3,  5, 
pi.  6,  fig.  15,  and  pi.  7,  fig.  25).  This  layer  is  probably  ectoplasmic 
in  nature;  it  stains  very  heavily  either  with  iron  haemotoxylin  or 
Mallory's  connective  tissue  stain.  Even  in  well-stained  whole  mounts 
it  may  be  clearly  made  out.  In  fact  it  may  easily  be  mistaken  for  an 
external  structure,  it  shows  so  clearly.  From  the  alveolar  ectoplasm 
this  boundary  layer  is  separated  by  a  thin  layer  of  regularly  placed, 
small,  cubical  alveoli,  and  from  the  more  homogeneous  entoplasm  by 
a  similar  alveolar  layer  consisting  of  large,  more  definitely  cubical 
alveqji.  This  boundary  layer  with  its  two  investing  layers  of  alveoli 


68  University  of  California  Publications  in  Zoology        [VOL.  13 

forms  a  sort  of  sack  enclosing  the  entoplasm,  into  which  opens  the 
oesophagus  (oes.,  pi.  4,  fig.  3)  and  out  of  which  leads  the  rectum 
(rect.,  pi.  4,  fig  3).  This  "boundary  layer,"  as  above  described, 
refers  only  to  the  definite  structureless  membrane  separating  the 
ectoplasm  from  the  entoplasm  and  bounded  on  either  side  by  a  layer 
of  alveoli.  Eberlein  (1895,  p.  244,  245)  describes  for  this  boundary 
layer  ("Grenzschicht")  a  definite  fibrillar  structure  and  is  inclined 
to  assign  to  it  also  two  or  more  alveolar  layers.  Giinther  (1900,  p. 
643)  states  emphatically  that,  notwithstanding  Eberlein 's  description, 
he  is  unable  to  find  any  alveolar  structure  in  the  above-named  layer. 
It  must  be  noted,  however,  that  the  oesophagus  contains  within  its 
walls,  as  will  be  described  later,  many  fibrillae  which  are  in  this  paper 
designated  as  oesophageal  retractor  strands  (oes.  retr.  sir.,  figs.  B,  D ; 
pi.  4,  fig.  3)  and  that  in  certain  portions  of  the  body  the  oesophagus 
comes  to  lie  in  such  close  contact  with  the  boundary  layer  as  to  defy 
microscopic  separation  of  the  two.  Hence,  in  these  places,  it  is  easy 
to  see  how  a  fibrillar  structure  might  be  assigned  to  the  boundary 
layer.  Careful  investigation  of  this  point,  however,  indicates  an  essen- 
tially non-fibrillar  structure  for  the  true  boundary  layer. 

The  micronucleus  and  macronucleus,  and  the  two  contractile 
vacuoles,  lie  in  depressions  on  the  outside  of  this  sack.  At  the  anterior 
end  of  the  body  this  boundary  layer  dips  down,  approaches  the  oeso- 
phagus as  a  funnel-shaped  depression,  accompanies  it,  and  finally 
blends  with  the  incoming  cuticle  (pi.  4,  fig.  3).  In  the  same  way  it 
approaches  the  rectum,  and  ascends  with  it  to  the  caecum,  on  the  sides 
of  which  it  blends  with  the  cuticle  and  becomes  lost  upon  it.  That 
this  layer  belongs  to  the  ectoplasm  rather  than  to  the  entoplasm  is 
determined  from  the  following  facts.  (1)  AVhen  these  Protozoa  are 
surrounded  by  an  irritating  chemical  medium,  or  by  disturbing  physi- 
cal conditions,  the  entoplasm  frequently  flows  out  through  the  gullet, 
but  in  these  cases  the  boundary  layer  always  remains  with  the  ecto- 
plasm. (2)  While  D.  ecandatum  is  feeding  the  entoplasm  is  in  con- 
stant, definite  motion.  The  boundary  layer  takes  no  part  in  this 
movement  but  remains  stationary  with  the  ectoplasm.  (3)  After  feed- 
ing, the  entoplasm  shows  many  changes,  which,  however,  are  not  dis- 
tinguishable in  either  the  boundary  layer  or  in  the  ectoplasm. 

ENTOPLASM 

The  entoplasm  (ent.,  pi.  4,  figs.  3-5;  pi.  7,  figs.  25-33),  shows 
under  the  most  powerful  lenses  (2600-3400  magnifications)  no  definite 


1914J  Sharp:  Diplodinium  ecaudatum  69 

structures  with  the  exception  of  a  single,  or  possibly  a  double,  layer 
of  alveoli  adjacent  to  the  boundary  layer,  but  appears  to  be  a  more 
or  less  homogeneous  mass  containing  food  particles  surrounded  by 
food  vacuoles.  The  entoplasm  is  entirely  surrounded  by  the  sack-like 
boundary  layer,  is  kept  from  flowing  out  through  the  mouth,  under 
normal  conditions  by  the  constriction  of  the  oesophageal  walls,  and 
from  flowing  out  through  the  anus  by  the  boundary  layer  covering 
the  rectum  and  caecum.  Scattered  throughout  the  entoplasm  are  food 
particles  surrounded  always  by  a  food  vacuole  (fd.  vac.,  pi.  4,  figs. 
3-5 ;  pi.  6,  figs.  14-19  ;  pi.  7,  figs.  25-33) . 

Eberlein  (1895,  p.  244)  describes,  in  the  case  of  Ophryoscolex 
inermis,  "Waben"  of  the  entoplasm,  which  group  themselves  around 
and  enclose  in  a  regular  manner  the  bits  of  food  which  the  animal  has 
taken  in.  No  such  structure  is  present  in  D.  ecaudatum,  but  it  must 
be  noted  that,  while  in  the  case  of  0.  inermis  the  food  consists  almost 
wholly  of  cellulose  fragments,  in  the  case  of  D.  ecaudatum  and  all  of 
its  forms  the  food  consists  almost  entirely  of  bacteria.  Evidence  of  a 
vegetable  food  ingestion  in  D.  ecaudatum  appears  only  in  those  cases 
in  which  the  host  (ox)  has  been  fed  just  before  slaughtering.  After 
the  ox  has  fed  on  alfalfa  hay  the  entoplasm  of  D.  ecaudatum  contains, 
for  two  or  three  hours  only,  green  chloroplastids,  as  well  as  the  bac- 
teria, but  no  cellulose  fragments.  In  living  animals,  during  feeding 
periods,  a  definite  streaming  of  the  entoplasm  is  discernible.  This 
streaming  of  the  entoplasm  will  be  described  under  observations  on 
the  living  animals. 

ORGANS  OF  THE  BODY 

Macronucleus. — The  macronucleus  (mac.,  pi.  3,  figs.  1,  2;  pi.  4, 
figs.  3,  5;  pi.  7,  figs.  29,  33),  is  situated  in  the  ectoplasm  between  the 
boundary  layer  and  the  cuticle  on  the  right  dorsal  side  of  the  body. 
In  general  the  macronucleus  has  a  rather  constant  size,  shape,  and 
position  within  the  animal.  Its  anterior  end  is  the  larger,  is  some- 
what curved,  and  the  longitudinal  axis  is  slightly  twisted  from  left 
to  right.  The  size  of  the  macronucleus  is  deserving  of  special  mention. 
Its  longitudinal  dimension  is  equal  to  about  five-eighths  of  the  entire 
length  of  the  body,  and  its  transverse  diameter  throughout  the  greater 
part  of  its  length  is  about  one-fourth  that  of  the  cross-section  of  the 
body.  Its  anterior  end  is  large,  bluntly  rounded  off  and  situated  just 
internal  to  and  just  below  the  base  of  the  right  extremity  of  the  dorsal 
membranelle  zone.  It  curves  slightly  dorsad  and  towards  the  posterior 


70  University  of  California  Publications  in  Zoology        [VOL.  13 

end.  The  diameter  remains  fairly  constant  until  just  below  the  mid- 
portion,  whence  it  gradually  diminishes,  to  end  in  a  blunt  point  just 
below  and  to  the  right  of  the  posterior  contractile  vacuole  (post.  c.  v., 
pi.  4,  fig.  3).  At  the  mid-part  on  the  dorsal  side  of  the  macronucleus 
is  a  shallow  depression  in  which  the  micronucleus  is  held.  The  macro- 
nucleus  is  entirely  surrounded  by  a  definite  nuclear  membrane.  This 
membrane  conforms  to  the  shape  of  the  macronucleus  and  forms  for 
it  a  well-defined  capsule.  Between  the  membrane  and  the  macro- 
nucleus  proper  is  a  clear  space  in  which  no  structures  are  visible  even 
under  the  most  powerful  magnifications  (2800  to  3400  diameters). 
The  nuclear  membrane  is  enclosed  on  the  outside  by  ectoplasm,  the 
reticular  structure  of  which  is  somewhat  more  regularly  arranged 
over  the  membrane  (see  pi.  4,  figs.  3,  5).  The  position  of  the  macro- 
nucleus  within  the  body  is  absolutely  fixed.  No  evidence  of  mobility 
such  as  has  been  suggested  for  the  macronucleus  of  Dasytricha  by 
Schuberg  (1888),  or  of  changes  in  its  position  as  suggested  for  some 
species  of  Opkryoscolex  by  Eberlein  (1895),  and  Giinther  (1899),  has 
been  found  in  D.  ecandatum.  This  absolute  fixation  of  the  macro- 
nucleus  in  D.  ecaudatum  is  undoubtedly  brought  about  through  its 
close  relation  to  the  skeletal  structure,  to  the  right  edge  of  which 
it  appears  to  be  firmly  connected  (pi.  3,  fig.  1;  pi.  4,  fig.  5;  pi.  7,  figs. 
26-30).  The  skeletal  structure,  therefore,  in  this  place  functions  as 
a  supporting  structure  for  the  macronucleus.  The  microphotographs 
(figs.  27,  28)  show  this  especially  well.  It  is  desired  to  call  attention 
in  this  place  to  the  fact  that  a  careful  study  of  the  best  preparations 
gives  no  evidence  which  tends  to  show  that  there  is  any  direct  com- 
munication between  the  oesophageal  wall  and  the  nuclear  membrane 
or  that  the  macronucleus  has  any  special  supporting  structures  other 
than  the  right  edge  of  the  skeletal  structure  and  the  surrounding 
boundary  layer  and  ectoplasm.  The  microphotographs  (pi.  6,  fig.  13; 
pi.  7,  figs.  26-30,  33)  might  indicate  otherwise,  but  this  is  because  the 
sections  were  cut  somewhat  obliquely  and  hence  there  is  a  slight  super- 
imposition  of  some  of  the  structures.  Hence  it  can  be  emphatically 
stated  that  structures  homologous  with  the  "Kernstiele"  of  Schuberg 
(1888),  Eberlein  (1895),  and  Giinther  (1899)  are  not  present  in  D. 
ecaudatum.  The  macronucleus  itself  is  distinctly  granular.  After 
iron-alum  haematoxylin  stain  these  granules  stand  out  clearly  and 
distinctly  and  may,  in  thin  cross-sections,  be  counted  (mac.,  pi.  4,  figs. 
3  and  5).  The  estimated  total  number  (based  on  examination  of  three 
specimens)  was  approximately  25,000  granules.  Interesting  changes 


1914]  Sharp:  Diplodinium  ecaudatum  71 

in  the  macronucleus  in  its  preparation  for  and  during  division  will 
be  described  in  a  subsequent  paper. 

Micronucleus. — The  micronucleus  may  be  clearly  distinguished 
even  in  the  living  non-stained  animals.  Here  it  appears,  by  trans- 
mitted light,  as  a  shining  little  body  situated  in  a  depression  on  the 
dorsal  side  of  the  macronucleus  about  midway  between  the  anterior 
and  posterior  extremities.  In  the  living  condition  it  appears  finely 
granular  in  structure  and  refracts  light  more  strongly  than  does  the 
macronucleus.  In  the  stained  preparations  the  micronucleus  (mic.,  pi. 
3,  figs.  1,  2;  pi.  4,  figs.  3,  5;  pi.  7,  figs.  29,  33),  is  seen  to  be  oblately 
spheroidal  in  shape,  somewhat  flattened  dorso-ventrally,  with  its  long 
axis  placed  longitudinally.  The  micronucleus,  like  the  macronucleus, 
is  encapsuled  by  a  clear,  well-defined  membrane,  between  which  and 
the  nuclear  substance  is  a  structureless,  clear  space.  The  capsule  of 
the  micronucleus  is  firmly  held  in  position  by  suspensory  fibers  (susp. 
f.,  pi.  4,  fig.  3),  which  appear  to  arise  from  the  nuclear  membrane  of 
the  macronucleus  above  and  below  the  depression  for  the  micronucleus 
and  its  capsule.  After  iron-alum  haematoxylin  stain  the  micronucleus 
substance  shows  blue-black.  After  Mallory's  connective  tissue  stain 
the  micronucleus  shows  bright  red.  This  is  especially  interesting  when 
considered  in  connection  with  the  fact  that  the  micronucleus,  together 
with  some  peculiar  masses  and  fibers  which  are  to  be  described  later, 
takes  this  stain  in  the  same  intensity  and  they  are  the  only  structures 
which  do  show  this  peculiar,  bright  red.  Since  these  peculiar  masses 
and  fibers  are  intimately  connected  with  the  organs  of  locomotion,  and 
since  they  and  the  micronucleus  invariably  take  the  same  stains  in 
the  same  intensity  it  would  seem  that  some  relationship  (chemical  at 
least)  must  exist  between  the  motor  apparatus  and  the  micronucleus, 
in  which  case  the  micronucleus  might  properly  be  termed  a  kineto- 
nucleus.  This  belief  is  strengthened  by  the  fact  that  many  cases  have 
been  observed  by  me  in  Diplodinium  in  which  the  macronucleus  was 
engaged  in  dividing  up  into  definite  chromosomes,  a  function  which 
in  ciliates,  except  in  0  patina,  is  generally  supposed  to  be  limited  ex- 
clusively to  the  micronucleus.  In  other  words,  it  appears  that  the 
macronucleus  here  may  be  analogous  to  the  trophonucleus  of  try- 
panosomes  and  so  the  question  is  raised  as  to  whether  or  not  it  might 
be  held  to  contain  the  so-called  "generative  chromatin."  This  phe- 
nomenon will  receive  further  consideration  in  my  paper  on  conjuga- 
tion and  reproduction  in  Diplodinium  ecaudatum.  The  granules  of 
the  macronucleus  are  too  small  and  too  numerous  to  permit  even  an 


72  University  of  California  Publications  in  Zoology        [VOL.  13 

attempt  at  an  approximation  of  their  number.  Changes  in  the  micro- 
nucleus  in  preparation  for  and  during  division  will  also  be  described 
in  a  subsequent  paper. 

ORGANS  OF  LOCOMOTION 

It  has  already  been  pointed  out  that  one  of  the  main  characteristics 
of  the  genus  Diplodinium  is  the  presence  of  what  Schuberg  (1888  and 
1891),  Eberlein  (1895),  and  Giinther  (1899  and  1900)  have  termed  a 
second,  or  dorsal,  membranelle  zone,  and  what  Fiorentini  (1889)  calls 
a  transverse  crown  of  cilia.  In  Diplodinium  ecaudatum  the  cilia  of 
both  the  dorsal  and  adoral  zones  are  grouped  to  form  clumps  or  tufts 
of  cilia.  Normally  all  of  the  cilia  composing  each  tuft  adhere  closely, 
just  as  do  the  hairs  of  an  ordinary  camel's-hair  paint-brush  when 
moistened  so  as  to  form  a  flexible  pencil.  The  composition  of  these 
brush-like  tufts  of  cilia  will  be  more  thoroughly  considered  below,  but 
here  attention  is  called  to  the  structural  difference  between  these  ciliary 
brushes  and  true  membranelles,  i.e.,  "flapping  or  swinging  membranes 
formed  by  fusion  of  two  or  more  transverse  rows  of  cilia  implanted 
side  by  side  and  adhering  to  form  a  flat  membrane"  (Minchin,  1912, 
p.  55).  Each  ciliary  brush  is  a  perfectly  definite  unit,  both  struc- 
turally and  functionally,  and  although  structurally  these  ciliary 
brushes  resemble  cirri  more  closely  than  they  do  membranelles,  still 
from  the  point  of  view  of  homology  it  seems  best  to  retain  the  designa- 
tion membranelle,  and  hence  in  this  paper  each  such  tuft  of  cilia  is 
referred  to  as  a  membranelle.  Throughout  the  Ophryoscolecidae,  as  a 
matter  of  fact,  these  membranelles  have  the  form  of  brushes  and  may 
be  designated  as  brush  or  penicillate  membranelles  in  contradistinction 
to  those  found  elsewhere,  as  for  example  in  the  Vorticellidae,  in  which 
the  cilia  of  the  membranelle  are  arranged  in  the  form  of  a  plat$  of  one 
or  two  lines  of  cilia  fused  in  one  locomotor  unit  of  flattened  type. 

Those  of  the  dorsal  region  are  termed  dorsal  membranelles  and 
those  of  the  adoral  region,  adoral  membranelles.  The  complete  row 
of  dorsal  membranelles,  together  with  the  inner  and  outer  dorsal 
lips  and  intervening  furrows,  is  termed  the  dorsal  membranelle  zone 
and  likewise  the  row  of  adoral  membranelles  with  its  corresponding 
inner  and  outer  adoral  lips  and  furrows  is  designated  as  the  adoral 
membranelle  zone.  The  dorsal  and  adoral  membranelle  zones  form 
the  locomotor  apparatus  of  the  animal  and  since  these  two  zones  are 
not  continuous  the  locomotor  apparatus  may  be  said  to  consist  of  two 
component  parts,  a  dorsal  locomotor  apparatus  or  dorsal  membranelle 


1914]  Sharp:  Diplodinium  ecaudatum  73 

zone,  which  is  locomotor  only  in  function,  and  an  adoral  locomotor 
apparatus  or  adoral  membranelle  zone,  which  is  both  locomotor  and 
nutritive  in  function.  Considered  from  the  point  of  view  of  its 
probable  evolution,  this  adoral  row  of  membranelles  was  undoubtedly 
primarily  nutritive  in  function,  but  owing  to  developmental  changes 
which  have  probably  taken  place,  combined  with  the  increasing  neces- 
sity of  greater  speed  or  possibly  the  decreasing  necessity  of  food- 
getting,  this  nutritive  function  has  gradually  been  given  over  to  that 
of  locomotion,  so  that  at  the  present  time  the  function  of  this  adoral 
row  of  membranelles  may  be  regarded  as  primarily  locomotor  and 
secondarily  nutritive.  This  conclusion  has  been  reached  after  due 
consideration  of  the  morphological  position  of  the  adoral  membranelles 
combined  with  careful  observations  made  of  the  living,  active  animals. 
Such  observations  invariably  lead  one  to  the  conclusion  that  the  adoral 
row  of  membranelles  functions  mainly  as  an  organ  of  locomotion. 
And  in  this  connection  it  is  interesting  to  note  that,  of  all  the  different 
species  of  the  genus  Diplodinium,  the  species  ecaudatum  is  provided 
with  the  most  powerful  and  the  most  complicated  organs  of  locomotion. 

Dorsal  locomotor  apparatus. — The  dorsal  locomotor  apparatus,  or 
dorsal  membranelle  zone,  is  placed  transversely  at  the  very  anterior 
extremity  of  the  dorsal  surface  of  the  body  and  consists  of  an  outer 
and  inner  dorsal  lip  (o.  d.  lip  and  i.  d.  lip,  figs.  B,  C ;  pi.  4,  figs.  3-4), 
an  outer  and  inner  dorsal  furrow  (o.  d.  fur.  and  i.  d.  fur.,  figs.  B,  C; 
pi.  4,  figs  3,  4),  and  a  row  of  twenty-six  membranelles  (d.  m.,  figs.  B,  C ; 
pi.  4,  figs.  3,  4;  pi.  7,  figs.  23-25). 

Beginning  at  a  point  just  dorsal  to  and  slightly  posterior  to  the 
dorsal  extremity  of  the  base  of  the  left  skeletal  area  (I.  sk.  a.,  pi.  3,  fig. 
2)  the  dorsal  row  of  membranelles  extends  transversely  around  the 
anterior  extremity  of  the  dorsal  surface  to  the  right  side,  where  it 
makes  a  short  curve  anteriorly  to  end  just  dorsal  to  the  dorsal  ex- 
tremity of  the  base  of  the  right  skeletal  area  (r.  sk.  a.,  pi.  3,  fig.  1 ;  pi. 
4,  fig.  4) .  The  bases  of  these  membranelles  extend  down  into  an  inner 
dorsal  furrow  (i.  d.  fur.,  fig.  B)  and  are  there  enclosed  by  a  fold 
of  the  ectoplasm  and  cuticle  which  in  cross-section  resembles  the 
human  lower  lip  and  has  therefore  been  designated  as  the  inner  dorsal 
lip  (i.  d.  lip,  fig.  B;  pi.  4,  figs.  3,  4).  Outside  of  the  inner  dorsal  lip 
is  a  second  fold  of  ectoplasm  and  cuticle,  not  so  high  as  the  inner  lip 
but  much  thicker  and  more  substantial.  This  outer  fold  also  resembles 
a  lip  and  is  therefore  termed  the  outer  dorsal  lip  (o.  d.  lip,  fig.  B ;  pi.  4, 
figs.  3#  4) .  The  outer  surface  of  the  outer  dorsal  lip  is  continuous 


74  University  of  California  Publications  in  Zoology        [VOL.  13 

with  the  surface  of  the  body  and  the  bases  and  extremities  of  both  lips 
are  continuous  with  the  ectoplasm  of  the  body.  Internal  to  the  inner 
lip  and  between  the  inner  and  outer  lips  are  deep  furrows  termed 
respectively  the  inner  and  outer  dorsal  furrows  (i.  d.  fur.  and  o.  d.  fur., 
fig.  B;  pi.  4,  figs.  3,  4).  In  the  inner  furrow,  which  is  bounded  ex- 
ternally by  the  inner  dorsal  lip  and  internally  by  a  portion  of  the  an- 
terior end  of  the  body  to  be  described  later  as  the  dorsal  disk  (d.  disk, 
fig.  B;  pi.  4,  fig.  3)  are  to  be  found  the  bases  of  the  dorsal  mem- 
branelles.  These  dorsal  penicillate  membranelles,  strong  and  vigorous 
in  life,  measure  from  one-twelfth  to  one-eighth  of  the  entire  length 
of  the  body  and  number  regularly  twenty-six  or  twenty-seven.  Each 
membranelle  consists  of  from  fifty  to  seventy-five  cilia  and  has  the 
appearance  of  a  very  long,  fine  camel  's-hair  brush.  It  is  almost  certain 
that  a  part  of  the  ciliary  processes  composing  each  membranelle  springs 
from  the  ectoplasm  of  the  dorsal  disk.  These  origins  are  termed 
anterior  ciliary  roots  (ant.  cil.  r.,  pi.  4,  fig.  3).  The  larger  number 
of  these  ciliary  processes  have  their  origin  in  the  ectoplasm  posterior 
to  the  outer  dorsal  groove,  however,  and  these  are  designated  as  the 
posterior  ciliary  roots  (post.  cil.  r.,  pi.  4,  fig.  3). 

In  heavily  stained  sections  there  appear  at  the  junction  of  the 
anterior  and  posterior  roots  slight  enlargements,  which  by  iron-alum 
haematoxylin  are  stained  more  intensely  than  the  root  strands  and  by 
Mallory's  connective  tissue  stain  show  the  peculiar  bright  red  which 
is  characteristic  of  nerves  stained  by  this  method  in  amphibian  tissue. 
In  the  living  condition  the  cilia  of  each  membranelle  cling  together 
as  do  the  hairs  of  a  wet  camel's-hair  brush,  but  in  the  fixed  specimens 
which  have  been  washed  in  alcohol  these  cilia  may  fluff  out  as  do  the 
hairs  in  a  dry  brush.  In  many  cases  the  cilia  composing  each  brush 
appear  to  be  twisted  spirally  like  the  stripes  on  a  barber's  pole.  The 
motion  of  these  ciliary  brushes  or  penicillate  membranelles  will  be 
discussed  later  under  observations  on  living  material. 

Adoral  locomotor  apparatus. — The  adoral  locomotor  apparatus,  or 
adoral  membranelle  zone  (ador.m.,  figs.  A,  B,  C ;  ador.m.z.,  pi.  3, 
fig.  2;  pi.  7,  figs.  20-22,  33)  is  much  the  more  complicated  of  the 
two  zones.  This  apparatus,  in  so  far  as  I  am  able  to  determine,  has 
never  been  correctly  figured  nor  described  for  the  species  ecaudatum. 
Briefly,  the  adoral  locomotor  apparatus  consists  of  two  rows  or  circles 
of  cilia,  an  outer  circle  composed  of  larger,  heavier  membranelles 
along  which  the  wave  of  contraction  passes  from  left  to  right,  and  an 
inner  circlet  of  smaller,  finer  cilia  along  which  the  waves  pass  from 


1914]  Sharp:  Diplodinium  ecaudatum  75 

right  to  left.  Thus  these  two  circles,  although  continuous,  run  in 
opposite  directions.  In  general  the  arrangement  of  the  inner  and  outer 
lips  and  the  inner  and  outer  furrows  is  the  same  as  in  the  case  of  the 
dorsal  zone.  The  following  differences,  however,  are  to  be  noted. 
The  outer  lip  (o.  ador.  Up,  figs.  A,  B;  pi.  3,  figs.  1,  2)  forms  an  almost 
complete  circle,  the  plane  of  which  is  not  horizontal  but  inclined  pos- 
teriorly on  the  left  side,  and  is  therefore  much  higher  on  the  right 
side.  This  lip  reminds  one  of  a  stiff  collar  which  is  a  little  higher 


D. 


—  ador. 


i.  ador.  lip J  ' o.  ador.  lip 


Fig.  A.  Anterior  view  of  the  oral  region  of  Diplodinium  ecaudatum  to  show 
peculiar  recurved  arrangement  of  adoral  membranelles  and  oral  cilia.  X  1150. 
ador.  m.,  adoral  membranelle ;  D.,  dorsal  side;  i.  ador.  lip,  inner  adoral  lip;  o.  ador. 
lip,  outer  adoral  lip;  or.,  oral  opening  or  cytostome;  or.  cil.,  oral  eilia;  or.  disk, 
oral  disk;  V.,  ventral  side;  X,  beginning  of  the  adoral  row  of  membranelles; 
Y,  the  point  at  which  the  adoral  row  of  membranelles  becomes  recurved  upon 
itself  to  form  the  row  of  oral  cilia. 


on  one  side  than  on  the  other  and  the  ends  of  which  do  not  quite  meet 
in  front — the  "front"  in  this  case  being  on  the  left  dorsal  side,  i.e., 
just-  above  the  left  extremity  of  the  dorsal  membranelle  zone.  The 
inner  adoral  lip  (i.  ador.  lip,  fig.  A;  pi.  3,  figs.  1,  2)  is  at  first  invisible, 
but  gradually  rises  above  the  edge  of  the  outer  lip  on  the  ventral  side, 
and  on  the  right  and  dorsal  sides  shows  prominently  above  the  outer 
lip.  This  inner  lip  might  also  be  likened  to  a  stiff  collar,  but  in  this 
case  the  two  ends  overlap,  the  terminal  end  passing  internal  to  the 
origin,,  where  it  may  end  gradually  and  indistinctly  as  in  Figure  A, 


76  University  of  California  Publications  in  Zoology        [VOL.  13 

or  it  may  be  continuous  with  the  oral  disk  as  in  figure  2,  plate  3,  or  it 
may  end  abruptly  as  in  figures  6  and  7,  plate  5,  all  depending  upon 
the  state  of  contraction  and  retraction  or  expansion  and  extrusion  of 
the  oral  region. 

The  adoral  membranelles  and  the  oral  tufts  of  cilia  are  very 
different,  both  in  their  composition  and  in  their  arrangement. 
Starting  from  a  point  (x,  fig.  A)  slightly  anterior. and  ventral  to  the 
left  extremity  of  the  dorsal  row  of  membranelles,  the  row  of  adoral 
membranelles  (ador.  m.,  figs.  A,  B)  circles  at  first  ventrally  and 
slightly  posteriorly,  then  to  the  right  and  slightly  anteriorly.  Still 
ascending,  it  next  curves  dorsally  and  then  to  the  left,  reaching  its 
highest  level  as  it  crosses  the  sagittal  plane.  Still  curving  to  the  left 
and  ventrally,  it  gradually  descends  to  a  more  posterior  level,  where  it 
occupies  a  position  internal  and  ventral  to  the  starting-point  and  on 
a  plane  about  midway  between  its  highest  and  lowest  levels.  At  this 
point  (y,  fig.  A)  the  adoral  zone  proper  may  be  said  to  terminate, 
but  the  row  of  membranelles  turns  suddenly  inward,  again  ascends  to 
a  higher  level,  then  recurving  upon  itself  begins  the  secondary  inner 
circlet,  which  runs  parallel  with,  but  in  a  direction  exactly  opposite 
to,  that  of  the  outer  row  of  adoral  membranelles.  At  the  sudden  turn 
(y,  fig.  A)  the  membranelles  become  much  shorter  and  finer,  lose  their 
brushlike  construction,  become  somewhat  flattened  in  appearance,  and 
may  now  be  described  as  a  row  of  less  distinct  groups  of  large  cilia 
surrounding  the  mouth  or  oral  opening,  viz.,  oral  cilia  (or.  cil.,  figs.  A, 
B,  C;  pi.  3,  figs.  1,  2;  pi.  4,  fig.  3).  The  membranelles  of  the  adoral 
zone  resemble  those  of  the  dorsal  zone  with  the  exception  that  they  are 
neither  so  long  nor  do  they  individually  contain  so  many  ciliary 
processes.  There  are  from  thirty  to  thirty-six  of  these  membranelles, 
each  consisting  of  from  forty  to  fifty  separate  cilia.  Their  bases  are 
situated  in  the  ectoplasm  immediately  posterior  to  the  inner  adoral 
furrow.  Here  also,  as  in  the  case  of  the  dorsal  membranelles,  each 
membrarielle  seem  to  be  composed  of  two  sets  of  roots,  which,  however, 
in  this  case  must  be  designated  as  internal  and  external  roots.  The 
internal  roots  take  their  origin  from  the  ectoplasm  in  the  region  of 
the  boundary  layer,  and  the  external  roots  take  their  origin  from  the 
ectoplasm  which  lies  close  to  the  outer  wall  of  the  body.  The  oral 
cilia  are  too  short  and  too  fine  and  are  located  too  centrally  to  be  of 
much  service  as  organs  of  locomotion,  but  since  they  are  a  direct  con- 
tinuation of  the  adoral  row  of  membranelles  they  will  be  described  in 
this  place. 


1914]  Sharp:  Diplodinium  ecaudatum  77 

Oral  cilia. — The  oral  cilia  (or.  cil.,  figs.  A,  B,  C;  pi.  3,  fig.  2;  pi.  4, 
fig.  3)  are  not  only  exceedingly  fine  but  are  also  exceedingly  thick, 
thus  making  it  difficult  to  arrive  at  any  very  definite  conclusion  regard- 
ing their  size,  number  or  arrangement.  It  is  certain,  however,  that 
they  are  in  some  way  connected  with  the  same  motor  apparatus  as  is 
the  adoral  row  of  membranelles,  of  which  they  seem  to  be  the  direct 
continuation.  In  life  they  appear  as  minute  tufts  which  are  in  almost 
constant  motion.  They  completely  surround  the  oral  opening  and 
extend  down  into  the  oesophagus  for  a  short  distance.  They  appear 
to  have  only  one  set  of  roots,  which  lie  close  to  the  oesophageal  wall 
and  end  in  or  near  the  circumoesophageal  ring  (dr.  oes.  ring,  figs.  B, 
C;  pi.  4,  fig.  3;  pi.  6,  fig.  15;  pi.  7,  fig.  33). 

ORGANS  OF  FOOD-TAKING 

The  organs  of  food-taking,  ectoplasmic  in  nature,  embrace  a  cyt- 
ostome, mouth,  or  oral  opening  (or.,  figs.  A,  B,  C;  pi.  7,  figs.  20,  21), 
oral  cilia  (or.  cil.},  oral  disk  (or.  disk),  to  some  extent  the  adoral  mem- 
branelles (ador.  m.),  and  an  oesophagus  (oes.). 

Cytostome. — The  cytostome,  mouth  or  oral  opening  (or.,  figs.  A, 
B;  pi.  4,  fig.  3).  is  an  elliptical  aperture  almost  entirely  surrounded 
by  the  oral  cilia  and  located  at  the  very  anterior  extremity  of  the  body, 
close  to  the  ventral  side  and  inclined  somewhat  ventrally  and  to  the 
left.  The  structure  of  the  oral  cilia  and  adoral  membranelles  has 
been  described  above;  their  function  we  shall  refer  to  later.  The 
mouth  opens  directly  into  an  oesophagus. 

Oesophagus.— The  oesophagus  (oes.,  fig.  B;  pi.  4,  figs.  3-5;  pi. 
7.  figs.  20-33)  extends,  as  a  closed  tube,  from  the  mouth  opening  to  a 
point  slightly  below  the  level  of  the  anterior  extremity  of  the  macro- 
nucleus  (pi.  7,  fig.  27).  At  this  level  the  inner  wall  disappears  and 
the  oesophagus  descends  as  an  open  or  one-sided  tube  to  the  extreme 
posterior  limit  of  the  entoplasm  (pi.  7,  figs.  31,  32).  At  the  oral  end 
it  is  exceedingly  small  and  rather  irregularly  elliptical  in  cross-section, 
with  the  long  axis  of  the  ellipse  extending  transversely  from  right  to 
left.  It  gradually  grows  larger  as  it  descends  posteriorly  through  the 
ectoplasm  into  the  entoplasm,  and  swings  obliquely  to  the  right  in 
such  a  manner  that  its  ventral  or  outer  wall,  which  is  much  the  thicker 
of  the  two  walls,  approaches  and  finally  comes  to  lie  next  to  the  right 
side  of  the  body  (oes.,  pi.  4,  figs.  4,  5;  pi.  7,  figs.  23-30).  Soon  after 
entering  the  entoplasm  the  dorsal  or  inner  wall,  which,  owing  to  the 
obliojue  descent  of  the  oesophagus,  is  now  turned  toward  the  left,  dis- 


78  University  of  California  Publications  in  Zoology        [VOL.  13 

appears,  and  so  leaves  the  left  or  internal  side  of  the  oesophagus  in 
free  communication  with  the  entoplasm  (oes.,  pi.  4,  figs.  3-5;  pi.  7, 
figs.  28-30).  The  walls  of  the  oesophagus  show,  according  to  Eberlein 
(1895,  pp.  245  and  255)  three  layers  ("Gewebsschichten")  :  (1)  an 
inner  layer  which  is  turned  toward  the  lumen  and  \vhich  is  a  thin 
continuation  of  the  cuticle;  (2)  the  middle  layer,  which  is  formed 
by  the  ectoplasm  and  is  characterized  by  a  closer  and  more  regular 
formation  of  the  reticulum;  and  (3)  an  outer  layer  which  is  formed 
by  the  boundary  layer  previously  described.  The  definite  arrangement 
of  the  "  Gewebesschichten "  which  Eberlein  (1895)  describes  for  the 
"Schlund"  of  Ophryoscolex  inermis  and  refers  to  as  being  the  same 
for  Diplodinium  maggii  (p.  255),  I  am  sorry  to  say,  I  cannot  confirm 
for  D.  ecaudatum.  The  oesophagus,  as  is  shown  in  longitudinal  sec- 
tions, does  not  come  into  contact  with  this  third  or  boundary  layer 
until  it  has  descended  some  distance  through  the  ectoplasm  (oes.,  pi.  4, 
fig.  3;  pi.  6,  fig.  15).  According  to  my  observations,  the  walls  of  the 
oesophagus  are  composed  (1)  of  a  thin  cuticular  continuation  from  the 
cuticle  of  the  body  (pi.  4,  fig.  3);  (2)  of  definite  longi- 
tudinal strands  (oes.  retr.  sir.,  fig.  B;  pi.  4,  figs.  3-5;  pi.  7, 
figs.  29,  33),  which,  since  they  are  attached  posteriorly  to  the  fused 
ventral  and  right  skeletal  structures,  wrould  seem,  both  from  their 
structural  connections  and  their  contractile  nature,  to  function  essen- 
tially as  retractor  strands;  (3)  of  certain  oesophageal  fibers  (oes.  f., 
fig.  B;  pi.  4,  fig.  4,  and  pi.  7,  figs.  23-25),  which  are  deemed  neural 
in  function  and  which  will  be  described  later;  and  (4)  of  the  ground 
substance  or  matrix  of  the  oesophagus,  ectoplasmic  in  nature  and  to 
which  or  in  which  the  above  structures  are  attached. 

The  number  of  these  oesophageal  retractor  strands  is  so  large, 
their  extent  so  great,  their  arrangement  so  complicated,  and  their  func- 
tion so  important  that  it  seems  best  to  give  them  a  further  and  more 
detailed  consideration.  Several  estimations  indicate  that  there  are 
from  100  to  150  of  these  retractor  strands  in  the  oesophageal  walls. 
They  appear  in  cross  sections  (oes.  retr.  sir.,  pi.  4,  fig.  5)  as  delicate 
radial  lines  joining  the  inner  and  outer  lamellae  of  the  oesophageal 
wall  and  in  longitudinal  sections  as  little  ribbon-like  bands  which 
extend  from  the  oral  opening  to  the  extreme  posterior  limit  of  the  ento- 
plasm. In  fact  it  seems  probable  that  a  number  of  these  strands  end 
in  the  vicinity  of  the  anus.  A  satisfactory  analysis  of  their  arrange- 
ment has  been  made  possible  through  a  comparative  study  of  the 
oesophageal  retractor  strands  in  Diplodinium  bursa,  which  is  a  some- 


Sharp:  Diplodinium  ecaudatum  79 

what  larger  form  and  one,  moreover,  in  which  these  oesophageal  struc- 
tures are  especially  clear. 

As  the  ventral  or  external  wall  of  the  oesophagus  comes  to  lie 
against  the  skeletal  structure  (pi.  7,  figs.  28,  29)  it  is  separated  from 
it  only  by  the  boundary  layer,  and,  as  the  inner  wall  disappears,  or, 
more  accurately  speaking,  separates  along  its  mid-line  and  its  sides 
also  become  flattened  against  the  boundary  layer,  it  becomes  more 
and  more  difficult  to  distinguish  the  latter  from  the  oesophageal  wall. 
As  this  oesophageal  wall,  if  such  it  may  still  be  called,  approaches  the 
posterior  end  it  comes  to  extend  over  more  and  more  of  the  circum- 
ference of  the  boundary  layer  until  (compare  figures  29  and  32,  plate 
7)  at  the  posterior  extremity  it  seems  to  completely  encircle  the  ento- 
plasm  and  is  distinct  from  the  boundary  layer  only  in  the  region 
of  the  rectum  which  seems  to  pass  down  between  the  two  (pi.  7,  figs. 
31,  32).  The  manner  in  which  the  rectal  sheath  is  formed  will  be 
considered  below.  It  will  suffice  here  to  note  that  it  contains  fibrillae 
(red.  f.,  pi.  4,  fig.  3)  which  appear  to  be  of  the  same  origin  and  take 
the  iron-alum  haematoxylin  stain  in  the  same  intensity  as  do  the 
oesophageal  rectractor  strands.  At  the  extreme  posterior  end  of  the 
entoplasm  which  is  just  dorsal  to  the  anal  opening  all  these  fibrillae 
or  retractor  strands  meet  in  a  point  which  is  probably  the  region  of 
final  constriction  at  the  time  of  division.  As  noted  above,  the  oeso- 
phageal wall  comes  to  lie  against  the  boundary  layer  and  the  skeletal 
structure  at  about  the  middle  of  the  body,  i.e.,  below  the  fusion  of  the 
right,  ventral,  and  left  skeletal  structures.  At  just  what  point  or 
points  the  oesophagus  is  attached  to  the  skeletal  structures  has  so  far 
defied  an  exact  determination,  but  that  such  attachments  are  made 
seems  altogether  certain. 

A  study  of  the  retracted  forms  (fig.  D)  justifies  this  belief  and 
also  furnishes  evidence  which  tends  to  prove  that  the  function  of  these 
oesophageal  strands  is  one  of  retraction.  This  conception  would  at 
least  explain  how  the  whole  oral  and  adoral  region  is  pulled  into  the 
body  when  the  organism  is  irritated.  Either  such  a  complicated 
oesophageal  structure  does  not  exist  in  the  heretofore  described  ciliates 
from  the  horse  and  the  ruminant,  or  it  has  been  overlooked  by  previous 
investigators. 

We  have  seen  that  in  the  normal,  active  condition  a  large  portion  of 
the  anterior  end  of  the  body  is  taken  up  with  the  organs  of  locomotion 
and  nutrition.  Three  other  structures,  however,  which  are  also  situated 
at  the>anterior  end  of  the  animal,  deserve  description.  To  these  struc- 


80  University  of  California  Publications  in  Zoology        [VOL.  13 

tures  we  have  given  the  names  dorsal  disk,  operculum,  and  oral  disk. 

Dorsal  disk. — The  dorsal  disk  (d.  disk,  fig.  B;  pi.  3,  figs.  1,  2;  pi.  6, 
fig.  15;  pi.  7,  fig.  33),  located  between  the  dorsal  membranelle  zone 
and  the  operculum  (op.,  fig.  B),  is  an  exceedingly  elastic  structure. 
Normally  it  has  the  shape  of  a  spherical  wedge,  resembling  one  of  the 
carpels  of  an  orange.  The  equator  corresponds  in  position  to  a  line 
drawn  from  the  middle  of  the  operculum  (op.}  to  the  middle  of  the 
dorsal  membranelle  zone,  and  the  axis,  or  diameter,  corresponds  to 
a  line  connecting  the  right  and  left  extremities  of  the  inner  dorsal 
lip  (i.  d.  lip).  The  size  and  shape  of  the  dorsal  disk  depend  entirely 
upon  the  degree  of  contraction  or  relaxation  of  the  operculum  and 
dorsal  membranelle  zone. 

Operculum. — The  operculum  (op.,  fig.  B;  pi.  3,  figs.  1-2;  pi.  7,  fig. 
33),  also  elastic  and  contractile  in  its  nature,  functions  as  a  protective 
structure  to  the  organs  of  locomotion  and  nutrition,  when  these  are 
retracted.  It  is  located  between  the  dorsal  disk  and  the  dorsal  curve 
of  the  outer  adoral  lip  and  is  attached  to  the  bases  of  the  right  and  left 
skeletal  areas  respectively  by  its  right  and  left  extremities,  which  thus 
serve  as  skeletal  attachments.  The  size  and  shape  of  the  operculum 
depend  largely  upon  its  state  of  contraction.  This  structure  will  be 
further  considered  under  observations  on  the  living  animals. 

Oral  disk. — The  oral  disk  (or.  disk,  figs.  A,  B;  pi.  4,  fig.  3;  pi.  7, 
figs.  20,  21),  resembles  the  dorsal  disk  only  slightly.  It  is  circular  in 
form  when  viewed  from  above  and  dome-shaped  when  viewed  from  the 
side.  The  oral  disk  almost  completely  surrounds  the  mouth  when  that 
organ  is  open  and  entirely  surrounds  -it  when  it  is  closed.  It  is  sur- 
rounded externally  by  the  adoral  membranelles  and  bounded  internally 
by  the  oral  cilia  which  separate  it  from  the  mouth  opening.  The  oral 
disk  is  thicker  on  the  right  side  than  on  the  left,  thus  throwing  the 
mouth  opening  rather  to  the  left  of  the  center  of  the  disk.  Tfie  func- 
tion of  this  disk  is  to  support  the  oral  cilia,  give  shape  to  the  mouth 
opening,  and  act  as  a  valve  to  close  the  oral  aperture. 

ORGANS  OF  DEFECATION 

The  organs  of  defecation  are  the  caecum  (caec.),  rectum  (rect.), 
and  anus  (an.). 

Caecum. — The  caecum  (caec.,  pi.  4,  fig.  3;  pi.  7,  fig.  33),  is  situated 
in  the  posterior  one-fourth  of  the  body  close  to  the  ventral  wall  and 
slightly  to  the  left  of  the  median  plane.  The  size  and  shape  of  the 
caecum  depend  entirely  upon  the  amount  of  excreta  which  it  contains. 


1914]  Sharp:  Diplodinium  ecaudatum  81 

When  empty  the  caecum  cannot  be  distinguished  in  the  living  animals, 
but  when  well  filled  it  may  be  discerned  as  a  round  or  balloon-shaped 
structure  whose  walls  become  more  and  more  definite  as  they  approach 
the  rectum.  We  noted  (p.  68)  under  the  description  of  the  boundary 
layer  that  in  the  stained  specimens  this  layer,  together  with  its  asso- 
ciated alveolar  layers,  is  lost  upon  the  sides  of  the  caecum.  The  caecum 
empties  directly  into  the  rectum. 

Rectum. — The  rectum  (rect.,  pi.  4,  fig.  3;  pi.  6,  figs.  16-19;  pi. 
7,  figs.  31-33),  short,  but  well  defined,  elliptical  in  cross-section,  leads 
from  the  caecum  to  the  cytopyge  or  anal  opening  (an.,  pi.  4,  fig.  3). 
In  the  case  of  the  rectum  the  three  layers,  i.e.,  (1)  cuticular,  (2) 
alveolar,  and  (3)  boundary  layer,  may  be  seen. 

It  is  intensely  interesting  to  note,  just  at  this  point,  that  during 
the  process  of  organ  formation,  in  the  predivision  stage,  the  new  oral 
cilia  form  in  a  little  cavity  which  is  situated  in  the  ectoplasm  between 
the  ventral  edge  of  the  oesophageal  wall  and  the  ventral  surface  of 
body  at  about  the  level  of  the  posterior  contractile  vacuole.  As 
development  progresses  this  ventral  edge  of  the  oesophageal  wall  is 
forced  more  and  more  toward  the  central  axis  of  the  body.  When 
division  is  completed  the  little  pocket  in  which  the  oral  region  of  the 
posterior  animal  was  developed  now  becomes  the  caecum  of  the  an- 
terior animal,  and  the  right  and  dorsal  wall  of  this  caecum  is  formed 
by  that  part  of  the  ventral  edge  of  the  oesophageal  wall  which  was 
pushed  in  by  the  developing  oral  cilia  as  described  above.  The  ventral 
wall  is  formed  by  the  boundary  layer.  This  becomes  all  the  more 
interesting  when,  as  will  be  described  under  observations  on  the  living 
animals,  it  will  be  noted  that  the  internal  posterior  current  (current 
No.  3)  of  the  entoplasm  is  directed  obliquely  from  the  left  ventral 
side  above  towards  the  right  ventral  side  below,  i.e.,  towards  the  most 
open  side  of  the  caecum.  Further  it  is  to  be  noted  that  lower  down 
the  whole  dorsal  wall  of  the  rectum  is  formed  by  what  was  once  the 
ventral  edge  of  the  oesophagus,  hence  this  wall  is  richly  supplied  with 
fibrillae  which,  moreover,  before  division  were  oesophageal  retractor 
strands.  That  these  strands  or  fibrillae  (rect.  f.,  pi.  4,  fig.  3)  have 
undergone  a  certain  amount  of  atrophy  or  degeneration  owing  to 
disuse,  or  possibly  a  change  in  function  is  indicated  by  the  fact  that 
with  Mallory's  modified  connective  tissue  stain  they  no  longer  stain 
as  distinctly  and  as  intensely  as  the  oesophageal  fibers  from  which 
they  were  derived.  After  iron  haematoxylin,  however,  they  are  quite 
distinct.  Whether  these  strands  still  retain  some  of  their  retractile 


82  University  of  California  Publications  in  Zoology        [VOL.  13 

nature  and  function  and  assist  in  the  process  of  defecation,  or  whether 
they  serve  merely  as  supporting  structures  to  the  rectum  it  is  not 
yet  possible  for  me  to  say. 

Anal  opening. — The  anal  opening  (an.,  pi.  4,  fig.  3;  pi.  6,  figs. 
18,  19;  pi.  7,  figs.  32,  33),  which  is  a  mere  slit,  is  located  at  the  pos- 
terior extremity  of  the  body  close  to  the  ventral  side. 

The  caecum,  rectum,  and  anus  have  to  do  with  the  discharge  of  the 
more  solid  particles,  i.e.,  the  undigested  remnants  of  the  bacteria  upon 
which  the  animal  feeds.  The  fluid  excreta  are  gotten  rid  of  by  means 
of  the  contractile  vacuoles. 

ORGANS  OF  EXCRETION 

These  are  the  contractile  vacuoles  (ant.  c.  v.  and  post.  c.  v.,  pi.  4, 
fig.  3;  pi.  6,  figs.  15,  17;  pi.  7,  figs.  28,  30),  which  are  also  ectoplasmic 
structures.  They  are  two  in  number  and  anterior  and  posterior  in 
location.  The  anterior  contractile  vacuole  (ant.  c.  v.,  pi.  4,  fig.  3),  is 
located  close  to  the  dorsal  wall  in  the  median  sagittal  plane  about  mid- 
way between  the  dorsal  membranelle  zone  and  the  micronucleus  and 
just  to  the  left  of  the  macronucleus.  The  posterior  contractile  vacuole 
(post.  c.  v.,  pi.  4,  fig.  3),  is  similarly  located  in  the  posterior  half  of  the 
body  close  to  the  mid-dorsal  wall,  half  way  between  the  micronucleus 
and  the  posterior  end  of  the  body  and  just  to  the  left  of  the  macronu- 
cleus. Each  vacuole  when  distended  is  ellipsoidal  in  shape,  slightly 
larger  than  the  micronucleus,  and  is  surrounded  by  a  slightly  differ- 
entiated ectoplasm  previously  described.  Each  vacuole  opens  on  the 
dorsal  surface  through  a  small  canaliculus  and  a  minute  pore  (ant.  c.  v., 
pi.  4,  fig.  3).  For  the  species  D.  ecaudatum  these  contractile  vacuoles 
have  proved  almost  absolutely  constant  both  in  number  and  in  position. 
In  fact,  out  of  the  large  number  of  individuals  of  this  specias  which 
have  been  observed  by  me  during  the  past  three  years,  not  more  than  a 
dozen  have  shown  the  single  vacuole  as  figured  by  Fiorentini  (1889, 
pp.  15,  16,  pi.  3,  figs.  1-2)  and  by  Eberlein  (1895,  pp.  262,  263,  pi.  18, 
figs.  18-19). 

NEUROMOTOR  APPARATUS 

We  now  come  to  the  description  of  what  is  believed  to  be  the  most 
interesting  structure  in  the  anatomy  of  this  organism,  a  structure  so 
intimately  and  so  peculiarly  connected  with  the  motile  parts  of  the 
body  that  its  function  as  a  neuromotor  apparatus  is  strongly  indicated. 


1914]  Sharp:  Diplodinium  ecaudatum  83 

Here  again  attention  is  called  to  the  fact  that  the  literature  on  these 
animals  is  devoid  of  any  suggestion  of  the  structure  which  is  here 
described.  The  possibility  of  this  structure  functioning  as  a  motor 
apparatus  or  even  possibly  as  a  neuromotor  apparatus  is  suggested  and 
for  purposes  of  description  the  designation  neuromotor  apparatus  will 
be  used,  and  its  constituent  parts  will  be  described  as  a  motorium  or 
motor  mass  (m.  m.,  figs.  B,  C;  pi.  4,  fig.  3;  pi.  6,  figs.  14-16;  pi.  7, 
figs.  21-24,  33),  a  dorsal  motor  strand  (d.  m.  sir.),  a  ventral  motor 
strand  (v.  m.  sir.),  opercular  fibers  (op.  /.),  an  adoral  lip  strand  (ador. 
lip  str.),  oesophageal  fibers  (oes.  /.),  and  a  circumoesophageal  ring 
(dr.  oes.  ring).  The  term  motorium,  as  applied  here,  is  used  in  its 
anatomical  or  neurological  sense,  i.e.,  the  common  center  of  motor 
influences.  It  may  be,  however,  that  we  have  here  a  condition  in  which 
nervous,  contractile,  and  supporting  elements  are  in  so  primitive  a 
stage  of  evolution  as  to  be  incapable  of  separation  into  purely  nervous, 
purely  contractile,  or  purely  supporting  structures.  The  structural 
conditions  and  the  observations  on  the  living  animal  in  activity,  how- 
ever, suggest  emphasis  upon  the  neural  rather  than  upon  the  con- 
tractile, or  supporting  nature  of  these  structures,  though  not  excluding 
the  latter  two.  They  are  probably  comparable  to  the  simple  fibres  of 
Stentor  figured  by  Neresheimer  (1903)  and  regarded  by  him  as 
' '  neurophanes. ' ' 

Motorium. — The  motorium  or  motor  mass  (m.  m.,  figs.  B,  C ;  pi.  4, 
fig.  3;  pi.  6,  figs.  14-16;  pi.  7,  fig.  33),  is  a  very  small  mass  of  chem- 
ically differentiated  tissue  located  rather  deeply  in  the  ectoplasm,  just 
above  the  base  of  the  left  skeletal  area  (1.  sk.  a.,  figs.  B,  C ;  pi.  4,  fig.  3) 
and  between  the  left  extremities  of  the  dorsal  and  adoral  membranelle 
zones.  This  motor  mass  was  first  noted  in  sections  stained  with  my 
modification  of  Mallory's  connective  tissue  stain.  It  was  discovered 
that  here,  in  the  region  just  described,  was  a  mass  of  tissue  which  had 
stained  rather  intensely  and  showed  by  transmitted  light  the  same 
bright  red  color  which  was  noted  in  the  case  of  the  micronucleus. 
Further  investigation  along  this  line  revealed  the  fact  that  not  only 
was  this  mass  constant  but  (1)  that  it  was  connected  dorsally,  by 
means  of  a  delicate  strand,  i.e.,  dorsal  motor  strand  (d.  m.  str.,  figs. 
B,  C;  pi.  4,  fig.  4),  with  the  bases  of  the  dorsal  membranelles,  also 
a  branch  strand  ran  along  the  base  of  the  inner  dorsal  lip,  i.e.,  the 
dorsal  lip  strand  (d.  Up  str.)  ;  (2)  that  a  fine  strand,  the  ventral  motor 
strand  (v.  m.  str.),  ran  from  it  to  the  bases  of  the  adoral  membranelles, 
alsoxthat  a  branch  strand  left  this  ventral  motor  strand  and  passed 


84 


University  of  California  Publications  in  Zoology        [VOL.  13 


along  the  base  of  the  inner  adoral  lip,  the  adoral  lip  strand  (ador.  lip 
str.),  and  that  many  well-defined  fibers  passed  from  it,  following  the 
contour  of  the  opereulum  towards  the  right  to  become  lost  in  the  im- 
mediate vicinity  of  the  base  of  the  right  skeletal  structure.  These  are 
the  opercular  fibers  (op.  /.).  Most  interesting  of  all,  however,  was 
the  apparently  perfectly  definite  connection  with  a  ring  of  substance 
surrounding  the  oesophagus  at  just  about  the  level  of  the  outer  adoral 
furrow.  This  ring,  which  is  designated  as  the  circumoesophageal  ring 
(circ.  oes.  ring,  figs.  B.  C;  pi.  4,  fig.  3;  pi.  6,  figs.  14-16;  pi.  7,  fig. 


ador.  m.  -  r 


—  i.  ador.  fur. 
~ — «'.  ador.  lip 
}  r —  o.  ador.  fur. 

!• o.  ador.  lip 

tn.  m. 


Fig.  B.  Diplodinium  ecaudatum.  Semi-diagrammatic  representation  of  an- 
terior half  to  show  arrangement  and  relations  of  neuromotor  apparatus.  X  1150. 
ador.  lip  str.,  adoral  lip  strand;  ador.  m.,  adoral  membranelles ;  ant.  c.  v.,  anterior 
contractile  vacuole;  dr.  oes.  r.,  circumoesophageal  ring;  D.,  dorsal  surface; 
d.  dislc,  dorsal  disk;  d.  m.,  dorsal  membranelles;  d.  m.  str.,  dorsal  motor  strand; 
i.  ador.  Up,  inner  adoral  lip;  i.  d.  fur.,  inner  dorsal  furrow;  i.  d.  lip,  inner  dorsal 
lip;  1.  sk.  a.,  left  skeletal  area;  mac.,  macronucleus;  mic.,  micronucleus;  m.  -m., 
motor  mass  or  motorium;  o.  ador.  -fur.,  outer  adoral  furrow;  o.  ador.  lip,  outer 
adoral  lip;  o.  d.  fur.,  outer  dorsal  furrow;  o.  d.  Up,  outer  dorsal  lip;  oes., 
oesophagus;  oes.  f.,  oesophageal  fibers  (neural  fibers)  ;  oes.  retr.  str.,  oesophasreal 
retractor  strands  (contractile  strands);  op.,  opereulum;  op.  f.,  opercular  fibers; 
or.,  oral  opening  (cytostome;  or.  cil.,  oral  cilia;  or.  disk,  oral  disk;  sk.  lam., 
skeletal  laminae;  V.,  ventral  surface;  v.  m.  str.,  ventral  motor  strand;  v.  sk.  a., 
ventral  skeletal  area. 


1914] 


Sharp:  Diplodinium  ecaudatum 


85 


33)  as  well  as  all  of  the  fibers  described  as  leaving  the  motorium, 
showed  in  all  regions  the  same  bright  red  color.  Other  fibers  also 
staining  bright  red  are  found  in  the  oesophageal  walls.  These  are 
called  oesophageal  fibers  (oes.  f.,  fig.  B;  pi.  4,  figs.  3,  4),  but  thus  far 
it  has  not  been  definitely  decided  whether  they  take  their  origin  from 
the  motorium  or  directly  from  the  circumoesophageal  ring,  probably 
the  latter,  however.  It  was  noted  in  the  description  of  the  oral  cilia 
that  the  root-fibers  of  these  cilia  end  either  in,  or  very  close  to,  this 


D. 


d.  m.  — 

o.  d.  lip -t 

d.  m.  sir.  — 


r.  sk.  a.-  — 


ador.  m.*-~- 

dr.  oes.  r.< •>  j 

dr.  oes.  r.  sir -> 

V. 


.  a. 
1  -  -  -    ador.  lip.  sir. 


Fig.  C.  Diplodinium  ecaudatum.  Diagrammatic  representation  of  the  moto- 
rium or  neuromotor  mass  and  its  neuromotor  strands  seen  from  the  anterior  end. 
X  1150.  ador.  lip  sir.,  adoral  lip  strand;  ador.  m.,  adoral  membranelles ;  dr.  oes., 
circumoesophageal  ring;  dr.  oes.  r.  sir.,  cireumoesophageal  ring  strand;  D.,  dorsal 
surface;  d.  lip  str.,  dorsal  lip  strand;  d.  m.,  dorsal  membranelles;  d.  m.  sir.,  dorsal 
motor  strand;  i.  d.  fur.,  inner  dorsal  furrow;  i.  d.  lip,  inner  dorsal  lip;  1.  sk.  a., 
left  skeletal  area;  m.  m.,  motorium  or  neuromotor  mass;  o.  d.  fiir.,  outer  dorsal 
furrow;  o.  d.  lip,  outer  dorsal  lip;  op.  f.,  opercular  fibers;  or.,  oral  opening  or 
cytostome;  r.  sic.  a.,  right  skeletal  area;  V.,  ventral  side;  v.  m.  str.,  ventral  motor 
strand. 


circumoesophageal  ring.     These  root-fibers  also  show  the  bright  red 
color  characteristic  of  wrhat  is  here  called  the  neuromotor  apparatus. 

In  particularly  well-stained  whole  mounts  (stained  with  iron-alum 
haematoxylin)  the  motorium,  the  dorsal  motor  strand,  the  ventral 
motor  strand,  and  the  oesophageal  ring  showr  very  clearly  Special 
attention  is  here  called  to  the  microphotographs  of  this  apparatus  (pi. 
6,  figs.  14-16;  pi.  7,  figs.  21-26,  33). 


86  University  of  California  Publications  in  Zoology        [VOL.  13 

The  facts  which  indicate  a  co-ordinating  (i.e.,  nervous)  function 
rather  than  a  contractile  or  a  supporting  function  for  the  above  de- 
scribed structure  may  be  summed  up  as  follows : 

1.  The  size,  shape,  position,  and  absence  of  direct  connection  with 
surrounding  structures  make  the  possibility  of  the  motorium  function- 
ing as  an  organ  either  of  contraction  or  of  support  seem  highly  im- 
probable.    For  in  order  to  function  as  an  organ   of  contraction  it 
would  necessarily  need  to  have  as  its  attachments  on  the  one  hand 
structures  which  are  fixed,  and  on  the  other  structures  which  are 
movable,  or  it  would  need  to  be  located  between  two  structures  both 
of  which  were  to  be  moved.     This  however,  is  not  the  case,  for  the 
motorium  seems  to  have  no  direct  connections  with  the  fixed  structures 
of  the  body,  nor  does  it  lie  in  the  direction  of  contraction  of  the 
oesophagus  and  oral  region,  which  upon  retraction,  descend  posteriorly 
into  the  body  (compare  figures  B  and  D).    Neither  would  the  assign- 
ment of  a  supporting  function  to  the  motorium  be  feasible,  first  because 
of  its  relatively  diminutive  size,  second  because  of  its  shape  which 
does  not  conform  particularly  to  that  portion  of  the  animal  in  which 
it  is  located,  and  third  because  of  its  location,  i.e.,  it  is  situated  in 
the  anterior  flexible  and  retractile  end  of  the  body  surrounded  by  the 
nonresistant,  semifluid  ectoplasm. 

2.  The  strands  which  leave  the  operculum  are  likewise  not   at- 
tached to  fixed  structures  but  lie  in  the  semifluid  ectoplasm  of  the 
opercular  region  and  in  the  inner  dorsal  and  inner  adoral  lips  which 
are  both  highly  mobile.     Also  it  is  to  be  remembered  that  both  the 
inner  dorsal  and  inner  adoral  lips  which  are  mobile  are  well  pro- 
tected and  well  supported  by  the  outer  dorsal  and  outer  adoral  lips 
respectively  both  of  which  are  fixed  and  rigid. 

3.  There  is  never  a  translation  of  the  parts  in  the  direction^  of  the 
strands  leaving  the  motorium,  but  rather  in  a  direction  at  right  angles 
to  the  course  of  the  fibers,  thus  militating  against  a  contractile  function 
for  the  fibers.     An  apparent  exception  to  this  general  statement  is 
to  be  found  in  the  case  of  the  oesophagus  with  its  oesophageal  fibers, 
but  here  it  is  to  be  noted;  first,  that  these  oesophageal  fibers  end  in 
the  vicinity  of  the  micronucleus  without  any  discoverable  connection 
with  a  fixed  structure,  and  second  that  the  oesophagus  is  richly  sup- 
plied with  another  set  of  fibers   (the  oesophageal  retractor  strands), 
which  do  not  take  the  red  stain,  and  which  are  apparently  attached  to 
the  skeletal  structure  as  described  above. 


1914J  Sharp:  Diplodinium  ecaudatum  87 

4.  Every  mobile  territory  is  supplied  by  strands  from  the  central 
mass   (motorium)   and  especially  are  the  bases  of  the  membranelles, 
both  dorsal  and  adoral,  well  supplied  by  these  fibers. 

5.  All  parts  connected  by  this  neuromotor  system  act  in  perfect 
co-ordination.     For  example,  on  being  surrounded  by  an  irritating 
medium,  mouth,  oesophagus,  and  oral  disk  are  retracted,  not  in  the 
direction  of  the  motor  strands,  but  posteriorly  into  the  body,  the 
inner  adoral  and  inner  dorsal  lips  are  shot  forward  completely  en- 
closing the  adoral  and  dorsal  membranelles   (fig.  D),  the  motion  of 
these  membranelles  and  of  the  oral  cilia  is  suspended,  and  for  the 
time  being,  the  animal  remains  in  an  apparently  inactive  condition. 
On  being  again  surrounded   by  a   favorable  medium,   however,   the 
cilia  once  more  take  up  their  activity,  even  while  retracted  within  the 
body.     Finally  the  oral  region  is  protruded,  the  inner  adoral  and 
inner  dorsal  lips  return  to  their  original  position,  and  the  animal 
exhibits  all  its  former  liveliness  of  motion.    Not  only  do  the  two  zones 
of  membranelles  act  in  unison  when  the  animal  is  swimming  freely, 
but  during  semi-quiescent  periods  as  when  the  animal  is  feeding  on 
a  mass  of  bacteria,  the  adoral  membranelles  and  oral  cilia  may  be 
particularly  active  while  the  dorsal  membranelles  remain  motionless 
or  only  slightly  active.     Or  again  only  a  few  of  these  membranelles 
may  be  active  while  the  others  are  entirely  motionless,  thus  suggesting 
the  innervation  of  each  single  membranelle  by  a  separate  fiber. 

Not  least  in  significance  for  this  conception,  is  the  fact  that  this 
neuromotor  apparatus  is  located  in  the  most  advantageous  position 
possible  to  function  as  a  center  of  motor  co-ordination  in  an  animal 
which  is  exceedingly  active,  exceedingly  sensitive,  and  exceedingly 
responsive  to  external  stimuli,  and  one,  moreover,  which  exhibits  a  high 
degree  of  selective  feeding.  Also  in  this  connection  it  is  to  be  noted 
that  that  portion  of  the  animal  which  first  comes  in  contact  with  new 
media,  viz.,  the  operculum,  is  the  most  richly  supplied  with  fibers 
radiating  from  the  motorium  (op.  /.,  figs.  B,  C,  D ;  pi.  6,  figs.  13-15). 

A  consideration  of  the  location  and  the  distribution  of  the  opercular 
fibers  suggests  the  interesting  question  of  the  possibility  of  these 
fibers  having  a  sensory  function.  Such  a  view  is  not  advanced  to  the 
exclusion  of  other  possible  functions.  If,  however,  these  opercular 
fibers  do  serve  as  conductors  of  sensory  impulses,  then  their  location 
in  the  most  anterior,  most  exposed  portion  of  the  body,  is  one  of 
advantage,  especially  when  the  membranelle  zones  are  enclosed.  Such 
S 


88 


University  of  California  Publications  in  Zoology        [VOL.  13 


a  view  helps  to  explain  not  only  the  number  and  distribution  of  the 
opercular  fibers,  but  also  the  fact  that  the  oral  cilia  and  the  mem- 
branelles  of  either  or  of  both  zones  may  be  set  in  motion  within  the 
body,  i.e.,  before  the  protrusion  of  the  oral  region  and  the  return  of 
the  inner  adoral  and  inner  dorsal  lips  to  their  normal  positions,  when- 
ever the  animal  is  again  surrounded  by  a  favorable  medium. 

Such  a  hypothesis  would  also  help  to  explain  the  sometimes  sudden 
retraction  of  the  membranelle  zones  when  the  animal  bumps  into  an 
obstruction  or  swims  into  an  irritating  medium,  and  upon  no  other 
hypothesis  can  all  these  phenomena  of  retraction,  protrusion  and  won- 
derful co-ordination  of  membranelles  and  membranelle  zones  be  so 
easily  and  so  satisfactorily  explained. 


I 1.  ador.  lip 

retr.or.cil. 1     I     . ador.  m. 


i.  ador.  Up , 


i c.  a  dor.  Up 


post.  oil.  r. 


_^ ant .  c.  v. 


oes.  retr.  str.  - 


mac. 


mic. 


Fig.  D.  Diplodinium  ecaudatum.  Eetracted  form  constructed  from  camera 
hicida  drawings.  X  1150.  ador.  m.,  adoral  membranelles;  ant.  c.  v.,  anterior 
contractile  vaeuole;  bd.  I.,  boundary  layer;  circ.  oes.  r.,  circumoesophageal  ring; 
D.,  dorsal  surface;  d.  m.,  dorsal  membranelles;  i.  ador.  lip,  inner  adoral  lip;  i.  d. 
lip,  inner  dorsal  lip;  I.  sk.  a.,  left  skeletal  area;  mac.,  macronucleus ;  mic.,  micro- 
nucleus;  o.  ador.  lip,  outer  adoral  lip;  o.  d.  lip,  outer  dorsal  lip;  oes.,  oesophagus; 
oes.  f.,  oesophageal  fibers  (neural  in  nature)  ;  oes.  retr.  str.,  oesophageal  retractor 
strands  (contractile  in  nature);  op.,  operculum;  op.  f.,  opercular  fibers  (nervous 
in  nature);  or.  disk,  oral  disk  (retracted);  post.  cil.  r.,  posterior  ciliary  roots; 
retr.  or.  cil.,  retracted  oral  cilia;  sk.  lam.,  skeletal  laminae;  V.,  ventral  surface. 


1914]  Sharp:  Diplodinium  ecaudatum  89 

RETRACTED  FORM 

A  detailed  description  of  the  mechanism  of  the  retraction  of  the 
oral  zone  and  the  resulting  encasement  of  the  adoral  and  dorsal  mem- 
branelles  will  be  given  later  in  the  account  of  observations  on  the  liv- 
ing material.  At  this  point  will  be  given  only  a  brief  description  of 
figure  D,  which  is  a  reconstruction  of  three  camera  lucida  drawings 
made  from  three  paramedian  sagittal  sections  each  five  microns  thick. 
The  noteworthy  points  may  be  summed  up  as  follows:  (1)  The  whole 
oral  region  is  retracted  within  the  body.  (2)  The  oral  cilia  come  to 
lie  within  the  oesophagus  (retr.  or.  cil.).  (3)  The  attachment  of  the 
boundary  layer  to  the  oesophagus  is  pulled  posteriorly  to  a  considerable 
extent,  thus  showing  that  both  the  point  of  .attachment  of  the  oseopha- 
geal  retractor  strands  (oes.  retr.  sir.)  and  the  region  of  actual  con- 
traction of  these  strands  is  below  the  point  of  junction  of  boundary 
layer  with  oesophageal  wall  (compare  with  fig.  B).  (4)  The  inner 
adoral  lips  (i.  ador.  lips)  and  the  inner  dorsal  lip  (i.  d.  lip)  are  ex- 
tended in  such  a  manner  as  to  become  direct  continuations  of  the  outer 
and  more  rigid  adoral  and  dorsal  lips  (o.  ador.  lip  and  o.  d.  lip)  re- 
spectively and  at  the  same  time  to  meet  in  the  epioral  line  in  the  case 
of  the  adoral  lips  and  to  meet  the  dorsal  edge  of  the  operculum  (op.) 
in  the  case  of  the  dorsal  lip,  thus  forming  a  complete  protective 
encasement  for  the  delicate  membranelles.  (5)  The  circumoesopha- 
geal  ring  (dr.  oes.  r.)  appears  to  be  somewhat  enlarged  and  stands 
out  even  more  clearly  than  in  the  extended  animals.  (6)  The  indi- 
vidual fibers  (oes.  /'.)  and  individual  retractor  strands  (oes.  retr.  str.) 
are  also  very  distinct  indicating  shortening  and  thickening.  This 
figure  does  not  show  the  motorium  and  its  connections  as  that  struc- 
ture lies  to  the  left  of  the  plane  here  depicted.  The  cut  ends  of  the 
opercular  fibers  (op.  /.),  however,  show  very  clearly.  In  the  descrip- 
tion of  the  neuromotor  apparatus  it  will  be  remembered,  strands  were 
described  which  passed  to  and  ran  along  in  the  inner  adoral  and  inner 
dorsal  lips  respectively.  These  strands  showed  fairly  well  in  those 
sections  stained  with  the  modified  Mallory's  connective  tissue  stain. 
No  sections  of  animals  in  the  retracted  condition,  however,  have  been 
prepared  with  the  Mallory  stain.  But  in  the  sections  of  the  retracted 
animals  stained  with  Heidenhain's  iron-alum  haematoxylin  no  trace 
of  such  strands  can  be  distinguished  within  the  extended  inner  lips. 
Whether  this  fact  is  due  to  the  extended  condition  of  the  lip  with  a 
corresponding  separation  of  the  fibers  so  as  to  make  them  too  minute 
for  identification,  or  whether  it  is  due  to  a  lack  of  "affinitv"  for  the 


90  University  of  California  Publications  in  Zoology        [VOL.  13 

haematoxylin  stain,  I  am  unable  to  say.  One  is  here  working  at  the 
limit  of  microscopical  vision  and  the  possibility  of  error  in  interpre- 
tation is  not  excluded  as  a  third  contingency. 


2.  Diplodinium  ecaudatum  forma  caudatum  Fiorentini 

PI.  5,  fig.  6 

Diplodinium  caudatum  Fiorentini   (1889),  pp.  15,  16,  pi.  3,  fig.  2. 
Diplodinium  rostratum,  Fiorentini  (1889),  p.  16,  pi.  3,  fig.  3. 
Diplodinium  rostratum,  Eberlein  (1895),  pp.  262-263,  pi.  18,   fig.  18. 

The  forma  caudatum  of  the  species  Diplodinium  ecaudatum  was 
first  described  and  figured  by  Fiorentini  (1889).  Although  he  gives 
only  a  very  brief  and  entirely  inadequate  description  of  the  shape 
and  structure  of  the  body  and  fails  to  interpret  correctly  the  things 
which  he  saw,  and  although  his  drawing  is  not  only  crude  and  inade- 
quate, but  also  in  some  respects  absolutely  erroneous,  still  he  was  the 
first  to  describe  this  form  and  in  many  ways  his  drawing  is  a  better 
representation  of  the  living  animal  than  is  that  of  Eberlein  (1895). 
It  is  not  desired  to  criticize  too  harshly  the  work  of  either  Fiorentini 
or  Eberlein,  but  merely  to  point  out  the  fact  that  the  previous  work 
on  this  form  is  entirely  inadequate. 

After  a  careful  study  of  the  description  and  figure  of  D.  rostratum 
by  Fiorentini  (1889)  it  seems  certain,  as  has  already  been  pointed 
out  (p.  51),  that  the  individual  which  Fiorentini  describes  as  D.  ros- 
tratum is  nothing  more  than  D.  e.  forma  caudatum  observed  shortly 
after  division. 

If  one  were  to  draw  the  anterior  portion  of  a  dividing  individual 
of  D.  ecaudatum  forma  caudatum  in  which  the  division  was  just  com- 
pleted, the  result  would  resemble  Fiorentini 's  figure  of  D.  rostratum. 
In  such  a  case  the  relatively  great  width  of  body,  the  blunt,  rpunded- 
off  posterior  end,  and  the  short,  stumpy  tail  would  all  be  accounted 
for.  Even  the  two  small,  unequal  contractile  vacuoles,  placed  close 
together,  are  characteristic  of  the  recently  divided  individual.  If 
this  interpretation  is  correct,  then  D.  rostratum  falls  into  the  synonymy 
of  D.  e.  forma  caudatum. 

Eberlein 's  (1895,  pp.  262-263)  description  of  Diplodinium  ros- 
tratum Fiorentini  covers  also  the  form  which  Fiorentini  describes  as 
Diplodinium  caudatum.  To  quote:  "Ferner  ist  das  von  dem  gleichen 
Forscher  in  seiner  Abhandlung  (1889)  auf  Taf.  Ill,  fig.  2,  abgebildete 
und  das  Diplodinium  caudatum  Fiorentini  beschriebene  Thier  zwei- 
felsohne  mit  dieser  Form  'identisch'  und  tauscht  nur  einzelne  Ver- 


1914]  Sharp:  Diplodinium  ecaudatum  91 

schiedenheiten  dadurch  vor,  dass  es  mehr  vom  Riicken  gesehen  darge- 
stellt  wurde. ' '  Hence  the  Diplodinium  rostratum  Fiorentini  described 
by  Eberlein  also  falls  into  the  synonymy  of  Diplodinium  ecaudatum 
forma  caudatum. 

The  action  of  Eberlein  (1895)  in  using  the  name  Diplodinium 
caudatum  for  a  new  species  discovered  by  him  has  already  been  dis- 
cussed. 

After  an  exhaustive  study  of  the  living  animals,  whole  mounts,  and 
transverse,  frontal,  sagittal,  and  oblique  sections  of  all  the  forms  of 
Diplodinium  ecaudatum,  both  from  the  originals,  camera  drawings,  and 
microphotographs,  it  is  certain  that  with  the  exceptions  of  normal 
variations  and  of  the  changes  in  form  and  structure  of  the  posterior 
extremity  occasioned  by  the  presence  of  one  or  more  spines, 'the  mor- 
phology of  all  of  these  forms  is  identical.  Hence  it  is  only  necessary 
in  this  place  to  consider  these  spines  and  the  changes  in  form  and 
structure  which  their  presence  occasions. 

Forma  caudatum  (pi.  5,  fig.  6)  appears  in  almost  as  great  num- 
bers as  does  forma  ecaudatum.  This  form  may  be  immediately  distin- 
guished from  all  the  other  forms  of  this  species  by  the  fact  that  a 
portion  of  the  posterior  end  of  the  body  is  prolonged  in  the  form  of  a 
tail-like  continuation,  or  spine  (sp.  1,  pi.  5,  fig.  6).  This  spine,  which 
is  designated  as  the  primary  spine,  takes  its  origin  from  the  whole  of 
that  portion  of  the  posterior  end  of  the  body  which  lies  ventral  to  the 
anal  opening.  Cross-sections  of  the  spine  at  or  near  its  base  appear 
bean-  or  kidney-shaped,  with  the  convex  margin  directed  ventrally 
and  the  concave  side  directed  dorsally ;  as  the  distal  extremity  is  ap- 
proached, the  cross-sections  become  more  nearly  circular.  Eberlein 
(1895,  p.  262)  describes  this  spine  as  being  "von  beiden  Seiten  etwas 
zusammengedrucktes, "  a  description  which  is  not  substantiated  by 
cross-sections  of  the  spine.  In  life  this  spine  is  equal  to  from  one- 
third  to  one-half  of  the  length  of  the  body  and  is  either  straight  or 
slightly  curved  distally  toward  the  dorsal  side.  In  fixed  specimens 
it  is  universally  more  or  less  curved  toward  the  dorsal  side.  The 
cuticle,  somewhat  thickened,  completely  covers  the  spine  and  the 
ectoplasm  is  prolonged  down  into  it.  In  no  other  way  does  the  presence 
of  this  spine  affect  the  morphology  of  the  animal. 

In  swimming  this  spine  seems  to  function  as  a  rudder,  for  it  is  to 
be  noticed  that  D.  e.  forma  caudatum  is  able  to  advance  in  a  more 
nearly  straight  line  than  is  D.  e.  forma  ecaudatum.  Dimensions  of  this 
form^are  given  below,  page  95. 


92  University  of  California  Publications  in  Zoology        [VOL.  13 

3.  Diplodinium  ecaudatum  forma  bicaudatum  forma  nova 

PI.  5,  fig.  7 

This  is  the  least  abundant  form  of  the  species.  It  is  characterized 
by  the  presence  of  a  secondary  spine  (sp.  2,  pi.  5,  fig.  7)  or  tail-like 
continuation  of  the  posterior  extremity  of  the  body.  The  base  of  this 
spine  is  located  to  the  left  of  the  median  plane  and  close  to  the  dorsal 
side  of  the  body  and  the  spine  itself  curves  ventrally  and  inward.  This 
secondary  spine  varies  in  size  from  a  mere  nodule  situated  just  to  the 
right  and  dorsal  to  the  anal  opening  up  to  a  spine  one-half  to  two- 
thirds  the  size  of  the  primary  spine.  The  morphology  of  the  secondary 
spine  is  identical  with  that  of  the  ventral  or  primary  spine.  The  pos- 
session of  a  secondary  spine  in  nowise  affects  the  position  or  mor- 
phology of  the  ventral  or  primary  spine,  but  does,  when  large,  affect 
to  some  extent  the  shape  of  the  dorsal  portion  of  the  posterior  end  of 
the  body.  When  more  than  one  spine  is  present  the  posterior  end  of 
the  body  is  relatively  enlarged  for  their  accommodation  and  the  body 
appears  more  nearly  cylindrical.  These  changes  in  size  relationships 
may  be  seen  by  a  comparison  of  figures  6-10,  plate  5.  In  no  other 
way  does  forma  bicaudatum  differ  from  forma  caudatum.  Dimensions 
of  this  form  are  given  on  page  95. 


4.  Diplodinium  ecaudatum  forma  tricaudatum  forma  nova 

PI.  5,  fig.  8 

This  also  a  relatively  rare  form,  is  distinguished  by  the  presence  of 
a  third  spine.  This  third  or  tertiary  spine  (sp.  3,  pi.  5,  fig.  8)  is 
located  rather  to  the  right  of  the  median  plane  and,  as  in  the  case  of 
the  secondary  spine,  curves  ventrally  and  inward.  The  tertiary  spine 
may  also  be  present  as  a  mere  nodule  situated  rather  close  to  the  right 
extremity  of  the  anal  slit  or  may  be  quite  as  large  as  the  secondary 
spine.  I  have  never  found  either  the  secondary  or  the  tertiary  spine 
to  be  as  large  as  the  primary  one,  although  the  secondary  and  tertiary 
sometimes  equal  each  other  in  size.  The  secondary  spine,  however,  is 
generally  the  larger  of  the  two. 

The  presence  of  this  third  spine  (sp.  3}  does  not  in  any  way  affect 
the  primary  (sp.  1)  and  secondary  spines  (sp.  2}.  The  posterior  end  of 
the  body,  however,  is  necessarily  a  little  larger. 

Dimensions  of  this  form  are  given  on  page  95. 


1914]  Sharp:  Diplodinium  ecaudatum  93 

5.  Diplodinium  ecaudatum  forma  quadricaudatum  forma  nova 

Pi.  5,  fig.  9 

This  is  again  a  very  abundant  form,  occurring  not  only  in  the 
majority  of  cattle  but  also  in  great  numbers  in  the  individual  hosts. 
In  fact  this  form  is  almost  as  abundant  as  is  D.  e.  forma  caudatum. 
This  form  is  characterized,  as  its  name  would  indicate,  by  the  pos- 
session of  four  spines  on  the  posterior  end  of  the  body.  This  fourth 
or  quaternary  spine  (sp.  4,  pi.  5,  fig.  9)  occurs  normally  on  the  right 
side  of  the  body  about  midway  between  the  primary  (sp.  1}  and  ter- 
tiary (sp.  2)  spines.  The  tertiary  spine  is  then  crowded  dorsally  until 
it  occupies  a  position  somewhat  more  dorsal  than  does  the  secondary 
spine  (sp.  2}.  In  about  four  per  cent  of  the  animals  examined  the 
quaternary  spine  (sp.  4)  was  located  on  the  left  side  between  the 
primary  (sp.  1}  and  secondary  (sp.  2}  spines.  When  this  is  the  case 
the  secondary  spine  (sp.  5)  is  crowded  so  far  dorsally  as  to  appear  to 
be  almost  exactly  opposite  to  the  ventral  or  primary  spine.  On  which- 
ever side  it  may  occur  this  quaternary  spine  (sp.  4)  is  almost  invari- 
ably the  smallest  and  ranges  in  size  from  a  mere  protuberance  to  a 
spine  almost  as  large  as  the  secondary  spine.  In  some  cases,  however, 
this  fourth  spine  was  even  larger  than  either  the  secondary  or  tertiary 
spines.  It  has  been  pointed  out  that  in  the  fixed  material  the  primary 
spine  ordinarily  curves  dorsally  while  the  secondary  and  tertiary 
spines  almost  invariably  curve  ventrally  and  inward.  In  the  case  of 
the  quaternary  spine  this  curvature  may  be  either  ventrally  and  in- 
ward, dorsally  and  inward,  or  merely  toward  the  main  axis.  It  is  to 
be  noted  that  the  designation  of  these  spines  as  primary,  secondary, 
etc.,  has  been  according  to  their  position  rather  than  to  their  size.  Up 
to  the  form  under  discussion  a  definite  relationship  has  seemed  to  exist 
between  size  and  position,  but  with  D.  e.  forma  quadricaudatum  this 
definite  relationship  no  longer  holds,  except  that  the  primary  spine 
is  always  the  largest,  and  the  first  three  spines  retain  their  relative 
positions.  In  this  connection  it  is  to  be  noted  that  considerable  varia- 
tion exists  as  to  the  relative  lengths  of  the  spines  both  in  relation  to 
each  other  and  in  relation  to  the  body.  That  is  to  say,  each  animal 
may  present  any  one  of  the  three  general  conditions:  (1)  all  of  the 
spines  may  be  short,  (2)  all  of  the  spines  may  be  long,  or  (3)  some 
of  the  spines  may  be  short  and  the  others  long.  Cross-sections  through 
the  bases  of  these  spines  (forma  quadricaudatum}  show  normally  a 
quack-angular  arrangement  and  in  many  cases  the  bases  of  the  sec- 


94  University  of  California  Publications  in  Zoology        tv°L- 13 

ondary,  tertiary,  and  quaternary  spines  appear  exactly  equal  in  size, 
with  the  primary  always,  however,  somewhat  larger.  Here  again  we 
note  the  enlargement  of  the  posterior  end  of  the  body  to  accommodate 
the  added  number  of  spines.  In  all  other  respects  this  forma  is  identi- 
cal with  D.  e.  forma  ecaudatum.  The  body  dimensions  are  given  on 
page  95. 


6.  Diplodinium  ecaudatum  forma  cattanei  Fiorentini 

Diplodinium  Cattanei  Fiorentini   (1889),  pp.  16-17,  pi.  3,  figs.  4,  5. 
PI.  5,  fig.  10 

This  form  is  not  very  abundant.  Not  only  is  its  presence  in  cattle 
the  exception,  but  even  when  present  it  occurs  only  in  small  numbers. 
The  distinguishing  feature  of  this  form  is  the  possession  of  five  pos- 
terior spines.  The  quintary  spine  (sp.  5)  occurs  on  the  right  side  just 
dorsal  to  the  primary  spine  and  curves  dorsally  and  inward.  In 
every  case  of  D.  e.  forma  cattanei  examined  the  arrangement  of 
spines  was  as  shown  in  the  figure  (pi.  5,  fig.  10),  i.e.,  a  very  large 
ventral  spine  (sp.  1),  a  large  secondary  spine  (sp.  2}  normally  placed, 
a  very  much  smaller  tertiary  spine  (sp.  3)  also  normally  located,  a 
very  broad,  somewhat  flattened  quaternary  spine  (sp.  4}  on  the  left 
side,  and  a  second  very  small  quintary  spine  (sp.  5)  on  the  right  side. 
In  every  case  the  two  spines  of  the  right  side  were  so  small  and  the 
two  spines  of  the  left  side  so  large  that  it  was  necessary  to  view  the 
spines  from  the  right  side  in  order  to  see  and  draw  the  small  spines. 
In  all  other  respects  the  morphology  of  the  spines  and  of  the  body 
was  what  would  be  expected.  The  dimensions  of  this  form  are  given 
below. 

Considerable  hesitation  was  at  first  experienced  in  assigning  Diplo- 
dinium cattanei,  as  described  and  pictured  by  Fiorentini  (1889),  to 
the  Diplodinium  ecaudatum  series.  As  a  matter  of  fact,  it  was  not  at 
all  certain  that  the  five-spined  form  described  by  this  investigator  was 
identical  with  the  one  occuring  in  my  material.  But  with  the  dis- 
covery of  all  the  intermediate  forms  of  the  series,  viz. :  the  two,  the 
three,  and  the  four-spined  forms,  coupled  with  the  fact,  on  the  one 
hand,  that  the  five-spined  form  which  was  present  in  my  material 
was  so  surely  a  member  of  the  ecaudatum  series,  and  on  the  other 
hand,  that  with  the  exception  of  the  number  of  vacuoles  pictured  for 
D.  cattanei  by  Fiorentini  (1889,  plate  3,  fig.  5),  my  five-spined  form 
corresponded  very  closely  to  his,  it  became  evident  that  the  two  were 


1914]  Sharp:  Diplodinium  ecaudatum  95 

identical.  D.  cattanei  was  therefore  the  end  member  of  the  Diplo- 
dinium ecaudatum  series  and  as  such  was  to  be  designated  as  D.  ecau- 
datum forma  cattanei.  This  conclusion  is  materially  strengthened 
by  the  fact  that  no  other  five-spined  Diplodinium  has  been  described, 
although  several  investigators  have  been  working  over  this  same  field 
since  Fiorentini's  (1889)  first  communication.  Apparent  discrepan- 
cies between  Fiorentini's  figures  and  descriptions  of  Diplodinium 
cattanei  and  my  figure  and  description  may  be  explained  upon  the 
assumption  that  one  of  his  figures  (pi.  3,  fig.  5)  and  his  description 
of  the  same  was  based  upon  an  abnormal  or  pathological  individual. 
The  possession  of  two  contractile  vacuoles  is  an  exceedingly  constant 
characteristic  of  this  species  and,  as  a  matter  of  fact,  in  his  figure  4, 
plate  3,  Fiorentini  pictures  only  two  such  vacuoles.  The  slight  differ- 
ence in  size  is  easily  accounted  for  when  it  is  remembered  that  the 
normal  variations  in  the  size  of  individuals  of  this  group  are  consider- 
able and  that  the  natural  tendency  of  the  observer  is  to  select  the 
larger  and  therefore  more  easily  figured  individuals. 

TABLE  OF  DIMENSIONS  FOR  ALL  FORMS  OF  Diplodinium  ecaudatum 
Five  animals  in  each  case 


Animals  ecaudatum 
measured      L.       W. 

caudatum        bicaudatum 
L.       W.            L.      W. 

tricaudatum 
L.      W. 

quadri- 
caudatum 
L.      W. 

cattanei 
L.      W. 

1st 

122 

40 

122 

45 

126 

44 

126 

46 

138 

52 

138 

53 

2nd 

132 

45 

126 

45 

122 

42 

122 

42 

112 

38 

126 

46 

3rd 

126 

44 

132 

48 

126 

45 

132 

45 

138 

54 

132 

52 

4th 

132 

45 

132 

50 

130 

48 

132 

48 

138 

54 

138 

54 

5th 

122 

43 

138 

53 

126 

45 

138 

45 

132 

50 

122 

44 

Average 

127 

43 

130 

48 

126 

45 

129 

47 

131 

50 

131 

50 

L. — Length  of  body  from  mouth  to  anus  in  microns. 
W. — Width  of  body  at  level  of  micronucleus  in  microns. 

These  measurements  were  taken  from  preparations  which  repre- 
sented the  average  size.  Exceptionally,  preparations  from  other 
stomachs  were  examined,  in  which  all  of  the  animals  were  either 
undersized  or  oversized,  thus  suggesting  the  occurrence  in  Diplodinium 
of  races  or  pure  lines  similar  to  those  described  by  Jennings  (1909) 
for  Paramecium. 

OBSERVATIONS  ON  THE  LIVING  MATERIAL 

One  of  the  first  things  that  the  observer  notices  when  studying 
these  interesting  little  animals  under  conditions  made  as  nearly  normal 
as  possible  is  the  terrific  rate  of  speed  at  which  they  travel.  Several 
observations  led  to  the  conclusion  that  at  the  normal  temperature, 


96  University  of  California  Publications  in  Zoology        [VOL.  13 

i.e.,  35?5  C,  an  individual  of  the  species  D.  ccaudatum  could  easily 
travel  a  distance  equal  to  twenty  times  its  own  length  in  less  than 
one  second.  It  must  be  admitted  that  no  accurate  measurements  were 
taken,  but  the  most  careful  and  conservative  estimates  led  to  the  above 
conclusion.  In  fact  we  are  convinced  that  this  species  holds  the  speed 
record  for  the  genus  Diplodinium  and  probably  for  all  of  the  genera 
described  thus  far  from  the  stomachs  of  ruminants.  The  normal 
course  taken  by  a  member  of  this  species  is  not  in  a  straight  line,  but, 
like  so  many  of  the  asymmetrical  protozoans,  it  advances  in  a  right 
spiral  as  does  the  point  of  a  corkscrew  when  penetrating  a  cork.  This 
fact  becomes  doubly  interesting  when  we  consider  the  build  of  the 
anterior  extremity  of  the  body.  We  noted  under  the  description  of 
the  organs  of  nutrition  that,  owing  to  the  greater  thickness  and  greater 
height  of  the  adoral  membranelle  zone  and  oral  disk  on  the  right  and 
dorsal  sides  of  the  mouth  than  on  the  left  and  ventral  sides,  the  plane 
of  the  mouth  was  directed  toward  the  left  and  ventrally.  Thus  we 
see  that  by  the  clockwise  rotation  and  the  spiral  course  of  the  body 
the  mouth  opening  is  brought  into  contact  with  a  greater  amount  of 
the  surrounding  medium  and  more  directly  than  could  possibly  be 
accomplished  in  any  other  manner  of  locomotion.  Keeping  in  mind 
that  all  the  evidence  points  toward  a  bacterial  diet  for  this  species, 
and  therefore  the  probable  necessity  of  great  numbers  of  these  small 
food  particles,  we  are  struck  with  the  wonderful  co-ordination  of 
locomotor  and  nutritive  organs,  which  makes  for  efficiency  in  food 
getting. 

Another  interesting  fact  was  one  day  forcibly  brought  to  my 
attention  when,  after  returning  to  the  laboratory  with  samples  from 
the  contents  of  ten  stomachs,  I  was  absolutely  unable  to  find  a  single 
member  of  the  species  D.  ecaudatum.  As  all  of  these  samples  had 
been  taken  from  the  same  herd  of  cattle,  the  question  arose,  Does 
geographical  environment  play  any  part  in  infecting  cattle  with  this 
protozoan?  Careful  records  kept  from  that  time  on  have  furnished 
the  following  information:  (1)  In  the  same  herd  some  cattle  may  be 
heavily  infected  with  ciliated  protozoans,  others  very  slightly.  (2)  In 
the  same  herd  some  cattle  may  be  heavily  infected  with  some  or  all 
of  the  species  of  one  genus  and  not  with  another,  while  other  cattle 
reverse  the  conditions  and  contain  heavy  infections  of  those  species 
and  genera  which  the  first  cattle  lacked.  (3)  In  the  same  herd  some 
cattle  contain  only  certain  forms  of  a  species,  while  other  cattle  contain 
only  other  forms  of  the  same  species.  (4)  In  the  case  of  Diplodinium 


19141  Sharp:  Diplodinium  ccaudatum  97 

ecaudatum  in  any  one  stomach  certain  groupings  of  forms  seemed  to 
be  the  rule ;  that  is  to  say,  we  have  here  a  general  condition  in  which 
(a)  forma  ecaudatum  may  be  the  only  form  present,  (6)  forma  can- 
datum  may  be  the  only  form  present,  (c)  forma  ecaudatum  and  forma 
caudatum  may  be  present  in  about  equal  numbers,  (d)  forma  quadri- 
caudatum  may  be  the  only  form  present,  (e)  forma  quadricaudatum 
may  be  associated  with  any  or  all  of  the  other  forms.  In  other  words, 
almost  any  combination  may  exist,  with  this  exception — the  forms 
bicaudatum,  tricaudatum,  and  cattanei  have  never  been  found  except 
in  the  presence  of  the  forma  quadricaudatum. 

As  noted  above,  under  technique,  satisfactory  observations  of  the 
activities  of  the  living  animals  could  be  obtained  only  by  reducing  the 
temperature  a  few  degrees,  which  reduction  and  control  was  made 
possible  by  the  automatic  constant-temperature  oven  to  which  refer- 
ence has  been  made.  In  this  oven  at  a  temperature  of  about  30°  C., 
the  following  observations  were  made,  for  at  that  temperature,  al- 
though only  5?5  C.  below  normal,  the  restless  activities  of  the  mem- 
bers of  this  species  are  slowed  down  sufficiently  to  permit  of  satis- 
factory study. 

The  cuticle  shows  clear  and  transparent,  the  skeletal  areas  are 
easily  defined;  in  fact  these  areas  are  much  more  plainly  seen  in  the 
living  animals  than  in  the  fixBd  material.  The  boundary  layer  be- 
tween ectoplasm  and  entoplasm  is  very  clearly  marked  off,  and  at 
certain  levels  the  macronucleus  and  the  micronucleus  can  be  easily 
distinguished,  the  macronucleus  having  the  characteristic  granular 
appearance  and  the  micronucleus  appearing  as  a  bright,  shining  little 
body,  refracting  the  light  strongly.  The  contractile  vacuoles  show 
up  much  more  clearly  in  the  living  animals  than  they  do  in  the  fixed 
material.  These  contractile  vacuoles  do  not  contract  suddenly  and 
disappear  as  in  the  case  of  Paramecium,  but,  on  the  contrary,  contract 
slowly  and  only  slightly,  then  gradually  enlarge  to  their  former  size. 
Their  action  is  more  of  a  true  pulsation.  The  caecum  and  rectum 
may  be  distinguished  just  before  and  during  the  process  of  defecation. 
In  a  few  cases  this  process  has  been  observed. 

The  streaming  of  the  entoplasm  referred  to  previously  may  usually 
be  observed  during  quiescent  periods  in  the  animal's  locomotor  activity. 
For  purpose  of  description  this  streaming  may  be  roughly  divided 
into  three  main  currents,  according  to  the  general  direction  assumed 
by  each:  (1)  a  peripheral  posterior  current,  (2)  an  anterior  current, 
anctr(3)  an  internal  posterior  current.  (1)  The  direction  of  the 


98  University  of  California  Publications  in  Zoology        [VOL.  13 

posterior  peripheral  current,  beginning  immediately  posterior  to  the 
level  at  which  the  oesophagus  passes  through  the  boundary  layer,  is 
obliquely  posterior  and  to  the  right,  i.e.,  following  the  general  direction 
of  the  oesophagus.  At  the  posterior  limit  of  the  sack  the  direction 
of  the  current  changes  so  as  to  flow  towards  the  left  dorsal  wall.  (2) 
The  anterior  current  beginning  at  this  point  follows  the  left  wall  of 
the  sack,  passing  obliquely  anteriorly  and  somewhat  ventrally,  i.e., 
in  exactly  the  opposite  direction  to  the  posterior  current.  When  the 
current  reaches  the  anterior  extremity  of  the  entoplasmic  sack,  which 
extremity,  as  will  be  remembered,  is  anterior  to  the  opening  of  the 
oesophagus  into  the  sack,  it  is  again  directed  posteriorly.  (3)  The 
internal  posterior  current  flowing  internally  to  the  first  described 
posterior  current  passes  posteriorly  to  the  region  of  the  caecum,  where 
it  becomes  lost  in  the  anterior  current.  Thus  we  see  that  the  two 
posterior  currents,  i.e.,  peripheral  and  internal,  pass  in  the  same 
direction  as  does  the  oesophagus,  and  so  may  assist  in  drawing  food 
particles  into  the  entoplasm ;  also  that  by  reason  of  its  flowing  directly 
toward  the  caecum  the  internal  current  may  assist  in  carrying  waste 
products  to  the  organ  of  defecation. 

Most  interesting  of  all  the  observations,  however,  were  those  upon 
the  action  of  the  dorsal  and  adoral  membranelle  zones  together  with 
that  of  the  operculum.  First  of  all,  it  was  noted  that  in  swimming 
the  organism  uses  both  zones  of  membranelles  and  that  normally  the 
contractions  take  place  as  waves  passing  from  one  extremity  to  the 
other.  In  the  case  of  the  dorsal  row  of  membranelles  these  waves 
usually  started  at  the  left  extremity  in  the  following  manner :  The 
first  membranelle  is  made  to  circumscribe  a  conical  space,  the  base 
of  which  corresponds  to  the  distal  extremity  of  the  membranelle  and 
the  apex  of  which  corresponds  to  its  attachment  to  the  bo(|y,  i.e., 
circumduction.  The  direction  of  this  movement  is  clockwise  and  the 
movement  has  no  sooner  started  in  the  first  membranelle  than  it  is 
begun  in  the  second,  and  so  on.  Even  when  the  animal  is  swimming 
slowly  a  second  wave  may  be  started  before  the  first  wave  has  reached 
the  opposite  extremity.  Thus  two  or  three  waves  of  contraction  may 
be  passing  along  the  row  of  membranelles  at  the  same  time.  In  the 
case  of  the  adoral  membranelles  the  movements  are  made  out  with 
much  more  difficulty  and  it  is  only  when  the  adoral  region  faces  the 
observer  that  satisfactory  results  can  be  obtained.  It  is  sometimes 
possible  to  bring  the  animal  into  this  position  by  a  careful  manipu- 
lation of  the  cover  glass.  In  general  the  movements  of  the  adoral 


Sharp:  Diplodinium  ecaudatum  99 

membranelles  resemble  those  of  the  dorsal  zone.  Normally  the  wave 
of  contraction  starts  at  the  junction  of  the  heavier  adoral  membra- 
nelles with  the  finer  oral  cilia  and  passes  first  to  the  right  and  then 
ventrally  and  to  the  left,  to  end  at  the  left  extremity  of  the  adoral 
row  of  membranelles.  The  movement  of  the  individual  adoral  mem- 
branelle  is  the  same  as  in  the  case  of  the  dorsal  membranelles,  i.e.. 
circumduction. 

A  second  interesting  observation  was  to  the  effect  that  any  single 
membranelle  or  any  set  of  membranelles  of  either  zone  could  be  moved 
at  the  point  of  stimulation,  by  simple  contact,  or  even  independently 
of  any  apparent  stimulus,  and  this  without  disturbing  the  other  mem- 
branelles, either  of  the  same  zone  or  of  the  other  zone,  a  phenomenon 
which  reminds  the  observer  of  the  result  obtained  by  stimulating  a 
single  tentacle  or  set  of  tentacles  of  the  sea  anemone.  This  fact  leads 
to  the  belief  that  each  penicillate  membranelle  is  supplied  by  an 
individual  ' '  nerve  fiber. ' '  As  the  oral  cilia  have  never  been  observed 
in  a  quiescent  state,  it  is  impossible  to  be  certain  of  the  direction  of 
their  wave  contractions. 

When  the  temperature  drops  too  low  or  the  animal  is  otherwise 
irritated,  either  mechanically  or  chemically,  the  oral  region,  viz., 
the  oral  cilia,  the  oral  disk,  and  the  oral  opening,  is  retracted  pos- 
teriorly into  the  body.  Simultaneously  with  this  the  inner  adoral  and 
inner  dorsal  lips  are  extended  in  such  a  manner  as  to  become  directly 
continuous  with  the  outer  adoral  and  outer  dorsal  lips  respectively, 
that  is  to  say,  the  outer  furrows  are  obliterated  and  the  two  lips  are 
smoothly  continuous  one  with  the  other.  When  the  oral  region  is  sud- 
denly retracted  the  popping  out  of  these  inner  lips  reminds  one  of 
the  popping  out  of  the  inverted  finger  tips  of  the  surgeon's  rubber 
glove  when  everted  by  air  pressure.  And  a  similar  explanation  is 
applicable  to  both.  In  other  words,  the  protrusion  of  the  inner  lips 
is  a  mechanical  occurrence  brought  about  through  the  contraction  of 
the  oesophageal  fibers,  which  pulls  the  oral  region  into  the  anterior 
end  of  the  body  and  thus  increases  the  pressure  in  the  semifluid 
ectoplasm.  According  to  the  laws  of  physics,  pressure  is  transmitted 
equally  in  all  directions.  Hence  when  the  increased  pressure  due  to 
the  inward  pull  of  the  oesophageal  retractor  strands  is  sufficient  to 
overcome  the  resistance  offered  by  the  weakest  portion  of  the  contain- 
ing wall  that  weakest  portion  will  yield  sufficiently  to  bring  again 
the  pressure  relations  to  an  equilibrium.  In  this  case  the  weakest 
portions  of  the  retaining  wall  are  the  inner  adoral  and  the  inner  dorsal 


100  University  of  California  Publications  in  Zoology        [VOL.  13 

lips  and  the  equilibrium  is  again  established,  in  the  one  case,  when 
the  different  portions  of  the  inner  adoral  lip  meet  in  the  epioral 
region,  and  in  the  other  case,  when  the  inner  dorsal  lip  meets  the 
dorsal  edge  of  the  operculum. 

If  it  can  be  assumed  that  these  inner  adoral  and  inner  dorsal  lips 
exhibit  any  degree  of  inherent  elasticity,  and  such  an  assumption  is 
entirely  within  the  bounds  of  probability,  then  an  explanation  of 
the  return  of  these  extended  inner  lips  to  their  normal  positions  to- 
gether with  the  simultaneous  protrusion  of  the  oral  region  is  also  to 
be  made  along  mechanical  lines  as  follows;  the  contraction  of  the 
oesophageal  fibers  is  relaxed  and  the  elasticity  of  the  inner  lips  and 
possibly  of  the  whole  anterior  end  of  the  animal,  i.e.,  the  tendency 
to  return  to  the  normal  position,  now  exerts  a  pressure  upon  the 
enclosed  ectoplasm  in  a  direction  opposite  to  that  which  caused  the 
protrusion  of  these  inner  lips,  with  the  result  that  as  the  inner  lips 
return  to  their  original  positions  the  oral  region  is  again  protruded 
and  the  animal  once  more  presents  the  normal  appearance  and  re- 
sumes its  activity. 

It  is  desired  in  this  place  to  call  attention  to  the  fact  that  previous 
observers  have  described  the  membranelle  zones  as  retractile  structures. 
Both  the  study  of  the  preparations  of  fixed  material  and  the  observa- 
tions on  the  living  animals  lead  to  the  conclusion  that  the  dorsal  mem- 
branelles,  and  probably  also  the  adoral  membranelles,  are  not  retracted 
within  the  body,  but  that  the  picture  presented  by  these  so-called  re- 
tracted forms  (fig.  D)  is  brought  about,  as  described  above,  by  the  pro- 
trusion of  the  lips.  In  other  words,  the  evidence  goes  to  show  that 
during  the  process  of  oesophageal  retraction  the  membranelles  remain 
stationary  and  become  encased  by  the  protrusion  of  the  inner  adoral 
and  dorsal  lips  respectively.  ^ 

In  watching  these  phenomena  of  retraction  and  expansion  in  the 
living,  active  animals  one  cannot  help  but  be  impressed  with  the  won- 
derful co-ordination  of  parts,  the  simple  and  yet  efficient  mechanism 
by  means  of  which  the  encasement  and  protection  of  the  delicate  mem- 
branelles is  effected  and  withal  the  probable  presence  of  at  least  the 
rudiments  of  a  nervous  system. 


1914]  Sharp:  Diplodinium  ecaudatum  101 

CONCLUSIONS 

1.  One  result  of  the  present  study  has  been  the  discovery  of  three 
new  forms   of  Diplodinium   ecaudatum,   namely  forma   bicaudatum 
(two  posterior  spines),  forma  tricaudatum   (three  posterior  spines), 
and   forma   quadricaudatum    (four   posterior   spines).      These   three 
forms,  together  with  D.  caudatum  Fiorentini   (one  posterior  spine), 
and  D.  cattanei  Fiorentini    (five  posterior  spines)   because  of  their 
structural  similarity  have  been  assigned  to  the  species  ecaudatum  (no 
posterior  spines). 

2.  Diplodinium  ecaudatum,  therefore,  consists  of  a  series  of  six 
forms  ranging  from  D.  ecaudatum,  without  posterior  spines,  up  to 
D.  cattanei,  a  form  with  five  posterior  spines.     No  other  structural 
characteristics  distinguish  these  forms  from  each  other. 

3.  The  reasons  for  assigning  Diplodinium  caudatum  and  the  forms 
bicaudatum,    tricaudatum,    quadricaudatum,    and    cattanei    to    the 
species   D.    ecaudatum   are   as   follows:      First,    with   the    exception 
of  the  presence  or  absence  of  the  spines,  the  dimensions  and  structures 
of  all  these  forms  are  practically  identical.     Second,  the  series  of 
spines  from  D.  e.  forma  ecaudatum,  without  spines,  up  to  D.  e.  forma 
cattanei,  with  five  spines,   is  complete.     Third,   with  the   exception 
of  the  primary  spine,  spines  of  all  sizes  are  to  be  found,  ranging  from 
mere  nodules  up  to  spines  which  are  equal  to  one-third  of  the  entire 
length  of  the  body. 

4.  The  reasons  why  each  of  these  types  of  this  organism  has  been 
designated  as  a  "forma"  according  to  the  number  of  spines  present 
are  as  follows : 

First— In  every  case  of  division  observed  animals  with  a  certain 
number  of  spines  gave  rise  to  two  daughter  animals,  each  of  which 
was  provided  with  the  original  number  of  spines.  Attention  is  called 
to  the  fact,  however,  that  none  of  these  cases  of  division,  so  far  as  the 
evidence  is  at  hand,  followed  at  once  after  conjugation,  and  that  it  is 
possible  that  division  immediately  following  conjugation  might  have 
resulted  differently. 

Second — That  the  presence  of  one  of  these  forms  in  the  stomach 
of  the  ox  in  no  wise  necessitates  the  presence  of  other  forms.  The  forms 
bicaudatum,  tricaudatum,  and  cattanei,  however,  have  never  been 
found  except  in  the  presence  of  forma  quadricaudatum. 

5.  The  genus  Diplodinium  has  been  revised,  as  a  result,  in  part, 
oMhe  discoverv  of  the  three  above-named  forms,  with  the  result  that 


102  University  of  California  Publications  in  Zoology        [VOL.  13 

the  number  of  valid  species  in  the  genus  has  been  reduced  from  ten 
to  five. 

6.  The  body  is  covered  by  a  very  resistant  cuticle,  divided  into 
definite  areas,  characterized  by  peculiar  surface  markings.     Three  of 
these  areas,  because  of  their  relation  to  underlying  skeletal  structures, 
are  designated  as  left,  ventral,  and  right  skeletal  areas.     These  three 
areas  with  their  underlying  skeletal  structures  are  separate  at  the 
anterior  end  of  the  animal,  but  merge  together  as  they  approach  the 
posterior  extremity.    They  afford  attachment  for  the  internal  retractor 
structures. 

7.  The  arrangement  of  the  oral  cilia  and  the  adoral  membranelles 
differs  from  that  previously  described  for  this  genus.     Starting  from 
a  point  on  the  left  side  of  the  animal,  near  to  the  anterior  extremity, 
the  adoral  row  of  membranelles  circles  from  left  to  right  around  the 
adoral  region  until  it  reaches  a  point  inside  of  and  opposite  to  that 
at  which  it  started,  then  turning  upon  itself  it  reverses  its  direction 
and  now  as  oral  cilia  circles  from  right  to  left  around  the  oral  opening. 

8.  There  is  present  in  D.  ecaudatum,  a  complicated  structure,  the 
neuromotor  apparatus,  which  is  probably  nervous  in  function.     This 
apparatus  consists  of  a  central  motor  mass  or  motorium,  from  which 
definite  strands  radiate:  one  to  the  roots  of  the  dorsal  membranelles 
(dorsal  motor  strand)  ;  one  to  the  roots  of  the  adoral  membranelles 
(ventral  motor  strand)  ;  one  to  the  circumoesophageal  ring  (circum- 
oesophageal  ring  strand)  ;  and  several  pass  out  into  the  ectoplasm  of 
the  operculum   (opercular  fibers).     Each  of  these  strands  may  send 
off  one  or  more  branches.    In  the  walls  of  the  oesophagus  both  nervous 
and  contractile  fibers  may  be  distinguished.    The  structural  and  func- 
tional relations  of  these  parts  are  such  as  to  indicate  that  they  con- 
stitute a  neuromotor  apparatus. 

The  Protozoa  have  often  been  defined  as  simple,  one-celled  animals. 
Calkins  (1909,  p.  1)  says  of  them:  "Their  beauty,  their  varied  modes 
of  life,  the  suddenness  of  their  appearance  and  disappearance,  the 
simplicity  of  their  structure  and  modes  of  reproduction  combine  to 
make  them,  even  to  the  superficial  observer,  a  fascinating  group." 
From  the  present  study  of  these  ciliated  protozoans  of  the  stomach  of 
the  ox  we  may  conclude  that  in  the  various  forms  of  the  species 
Diplodinium  ecaudatum  are  to  be  found  some  of  the  most  interesting 
and  also  the  most  complex  of  all  known  Protozoa. 

Transmitted  May  10,  1913. 


Sharp:  Diplodinium  ecaudatum  103 


ADDENDUM 

This  paper  was  accepted  for  publication  by  the  University  Press, 
May  10,  1913.  The  receipt  of  a  very  generous  gift.  May  22,  1913, 
made  possible  the  publication  of  plate  4  in  colors  and  also  the  addi- 
tion of  the  microphotographs,  plates  6  and  7.  The  preparation  of 
these  plates  has  delayed  publication. 

On  December  12,  1913,  after  this  paper  had  gone  to  press,  the 
Archiv  fiir  Protistenkunde,  of  November  11,  1913,  containing  Braune's 
excellent  paper,  ' ' Untersuchungen  tiber  die  im  Wiederkauermagen 
vorkommenden  Protozoen, "  was  received  at  this  laboratory.  Since 
Braune  has  worked  on  the  same  family,  the  Ophryoscolecidae,  as 
myself,  and  has  figured  and  described  for  Opkryoscolex  purkynjei 
Stein  structures  which  are  apparently  homologous  with  those  described 
by  me  for  Diplodinium  ecaudatum,  and  since  our  interpretations,  not 
only  of  the  morphology  but  also  of  the  functions  of  several  of  these 
structures,  differ  to  some  considerable  extent,  it  seems  necessary  to 
add  a  word  here.  It  must  be  kept  in  mind  that  although  our  observa- 
tions have  been  made  upon  somewhat  similar  organisms,  yet  notwith- 
standing their  close  relationship  these  organisms  may  present  many 
dissimilarities.  Having  made  no  comparative  study  of  the  minute 
structure  of  the  form  which  Braune  describes  I  must  content  myself 
with  a  brief  discussion  of  the  more  obvious  points  wherein  we  differ. 

Although  the  distinction  between  ectoplasm  and  entoplasm  may  be 
a  more  or  less  arbitrary  one  depending  upon  the  definitions  of  the 
observer,  still  the  separation  by  Braune  (1913,  p.  151,  and  pi.  6,  figs. 
38-41)  of  the  "  Ectoplasma, "  "  Grenzschicht, "  and  "Entoplasma" 
of  Schuberg  (1888),  Eberlein  (1895),  and  Giinther  (1899,  1900)  into 
his  so  called  "Entoplasma  a,"  " Fibrillenschicht, "  and  "Entoplasma 
b"  is  not  in  harmony  with  the  evidence  obtained  by  me  from  Diplo- 
dinium ecaudatum.  Nor  is  it  altogether  in  accord  with  Braune's  own 
work,  for  he  suggests  (p.  152)  the  correspondence  with  similar  layers 
in  Isotricha  prostoma  and  yet  in  this  form  both  in  his  description  (p. 
140)  and  in  his  figures  (pi.  5,  figs.  32,  33)  he  holds  that  the  ciliary 
roots  penetrate  only  as  far  as  the  "Grenzschicht"  which  separates 
ectoplasm  from  entoplasm  while  for  Opkryoscolex  purkynjei,  he  states 
(p.  158,  pi.  6,  fig.  37)  that  the  membranelle  roots  pass  through  not 
only  the  ectoplasm  and  ' '  Grenzschicht, ' '  but  also  penetrate  the  ' '  Ento- 
plasna  a." 


104  University  of  California  Publications  in  Zoology        [VOL.  13 

That  neither  his  description  of  the  anatomy  nor  his  interpretation 
of  the  function  of  the  ' ' Stiitzapparat "  of  Ophryoscolex  purkynjei 
will  hold,  even  in  the  main,  for  the  skeletal  structure  of  Diplodinium 
ecaudatum  may  easily  be  seen  from  a  glance  at  the  microphotographs 
(pi.  7,  figs.  20-33). 

Braune  (1913,  pp.  152-154,  and  pis.  6,  figs.  38-40)  states  in  the 
first  place  that  the  "Stiitzapparat"  is  a  unit  organization,  a  struc- 
ture ("einheitliches  Gebilde")  situated  in  the  "Entoplasma  a,"  and 
filling  the  entire  ventral  side.  Microphotographs  23-29,  plate  7,  show 
very  clearly  that  for  D.  ecaudatum  the  skeletal  structure  is  much 
more  complicated,  that  it  consists  of  three  component  parts,  well  de- 
fined at  the  anterior  end  (figs.  25-26)  and  merging  near  the  middle 
of  the  animal  (figs.  28-29)  ;  that  these  component  parts  are  situated 
in  the  ectoplasm,  and  that  at  the  anterior  end  of  the  animal  this 
structure  extends  over  the  entire  ventral  half  of  the  circumference 
of  the  body,  but  as  the  posterior  half  of  the  body  is  approached  the 
structure  comes  to  lie  more  and  more  to  the  right  side. 

A  second  point  made  by  Braune  is  that  the  "Stiitzapparat"  is  a 
plate  with  its  lateral  edges  bent  in  towards  the  inner  part  of  the  body 
and  its  anterior  ends  drawn  out  to  points  thus  better  to  surround  the 
oesophagus.  An  examination  of  the  microphotographs  22-29,  plate 
7,  shows  conclusively  that  in  the  case  of  D.  ecaudatum  the  skeletal 
structure  cannot  be  described  as  a  plate  with  its  lateral  edges  bent 
in  towards  the  inner  part  of  the  body  nor  are  the  anterior  ends  drawn 
out  to  points,  for  it  will  be  noted  that  figure  23,  plate  7.  which  repre- 
sents the  fifth  section  in  the  series,  and  one  which  is  the  most  anterior 
section  showing  this  skeletal  structure,  gives  absolute  evidence,  by 
measurement,  that  each  of  the  three  component  parts  is  actually  broader 
at  its  most  anterior  extremity  than  at  any  other  level  in  its^  entire 
length.  Also  in  these  first  five  or  six  sections  which  represent  the 
anterior  one  fourth  of  the  animal  it  is  plainly  evident  that  the 
oesophagus  has  no  definite  connection  with  the  skeletal  structure 
other  than  with  the  surrounding  ectoplasm. 

In  the  third  place  Braune  maintains  that  this  structure  may  be 
divided  into  three  layers,  (a)  an  outer  layer  composed  of  fine,  long, 
interlacing  fibrillae,  (b)  a  middle,  alveolar  layer  (described  by 
Giinther,  1899,  1900),  composed  of  very  large  alveoli  which  at  times 
occupy  the  whole  thickness  of  the  supporting  structure,  and  (c)  an 
inner  " Fibrillenlage "  situated  between  "Entoplasma  a"  and  "Ento- 
plasma  b"  which  exhibits  the  large,  parallel  fibrillae.  Again  exam- 


1914]  Sharp:  Diplodinium  ecaudatum  105 

ining  microphotographs,  figures  23-29,  plate  7,  it  will  be  seen  that  no 
fibrillar  layer  exists  between  the  skeletal  structure  and  the  cuticle 
nor  is  there  any  structure  which  might  correspond  to  the  "Fibril- 
lenlage. ' ' 

Braune  also  states  that  the  thin  right  edge  of  the  ' '  Stiitzapparat ' ' 
separates  the  macronucleus  from  the  "Entoplasma  b."  That  this 
does  not  hold  for  D.  ecaudatum  is  evidenced  by  figures  27-29,  plate  7. 

He  further  holds  that  the  importance  of  the  "Stiitzapparat"  lies 
in  its  relations  to  the  internal  structures  and  that  it  serves  as  a  sup- 
port not  only  for  the  gullet,  but  also  for  the  many  longitudinal  and 
transverse  fibrillae.  As  to  the  relation  between  skeletal  structure  and 
fibrillae  in  D.  ecaudatum  reference  will  be  made  later,  and  as  to  the 
skeletal  structures  serving  as  supports  for  the  oesophagus,  the  micro- 
photographs,  figures  20-23,  33,  show  that  the  oesophagus  extends 
20-24  microns  further  anteriorly  than  do  the  skeletal  structures,  that 
there  is  no  direct  connection  between  oesophagus  and  skeletal  struc- 
ture for  another  30-40  microns.  Microphotographs,  figures  28-30, 
however,  show  pretty  conclusively  that  soon  after  the  oesophagus  does 
become  attached  to  the  skeletal  structure,  this  latter  structure  dis- 
appears. In  other  words  the  skeletal  structure  serves  as  a  fixed  organ 
for  the  posterior  attachment  of  the  contractile  or  retractile  oesophageal 
strands.  Observations  made  upon  the  living  animals  and  examinations 
of  the  stained  sections  lead  to  the  conclusion  that  the  important  func- 
tions of  the  skeletal  structure  in  D.  ecaudatum  are  first  of  all,  to  give 
the  characteristic  shape  and  rigidity  to  the  body,  secondly  to  provide 
a  fixed  posterior  attachment  for  the  retractile  oesophagus  and  a  sub- 
stantial support  for  the  operculum  and  the  macronucleus,  and  thirdly 
by  a  combination  of  the  above  to  afford  protection  to  all  of  the  body 
structures. 

Braune 's  account  of  a  most  remarkable  network  of  fibrillae 
("Fibrillenapparat")  in  0.  purkynjei,  is  worthy  of  a  more  compre- 
hensive discussion  than  it  is  possible  to  give  it  here.  Only  the  more 
important  differences  between  this  "Fibrillenapparat"  and  the  neuro- 
motor  apparatus  described  for  D.  ecaudatum  will  be  discussed.  He 
says  (p.  156),  "Mit  dem  Nachweis  dieses  auBerordentlichen  Fibrillen- 
reichtums  ergibt  sich  aber  die  Schwierigkeit,  ihrer  in  der  Beschrei- 
bung  gerecht  zu  werden  und  von  ihrer  Schonheit  und  Harmonic  zu 
berechten, ' '  and  his  description  is,  in  the  main,  as  follows : 

1.  Separating  the  "Entoplasma  a"  from  the  "Entoplasma  b"  is 
a  saeli-like  "Fibrillenschicht  (Fig.  38,  Fb.  sch.)  "  which  is  an  "auBer- 


106  University  of  California  Publications  in  Zoology        [VOL.  13 

ordentlich  verzweigten  Fibrillensystem, "  but  which  represents  only  a 
part  of  the  complicated  "Fibrillenapparates"  and  is  to  be  regarded 
as  a  particular  structure  in  the  entoplasma.  These  "Fibrillen"  are 
longitudinal  and  are  internal  to  the  transverse  "Fibrillen"  next 
described. 

2.  The  whole  body  is  surrounded  by  a  large  number  of  almost 
parallel  transverse  "Fibrillen"  which  take  their  origin  from  one  edge 
of  the  ' ' Stiitzapparat "   (his  fig.  36,  qu.  fibr.)   and  pass  around  the 
dorsal  side  of  the  body  to  their  attachment  in  the  opposite  edge  of  the 
"Stiitzapparat."     They  assume  considerable  size  when  they  have  a 
particular  function  to  perform.     To  quote  (p.  158),  "So  finden  wir 
oberhalb,  der  Membranellenzone  zwei  quer  Fibrillenbiindel  (Fig.  36a, 
&),  die  in  einem  bestimmten  Abstand,  durch  mehr  oder  weniger  regel- 
maBig  angeordnete  Langsfibrillen  verbunden,  stehen. ' ' 

Although  the  same  fixing  and  staining  methods  (viz.,  Schaudinn's 
alcoholic  sublimate  solution  followed  by  Heidenhain's  iron-alum 
haematoxylin)  that  Braune  used  for  0.  purkynjei  have  been  used  on 
D.  ecaudatum  it  was  not  possible  to  demonstrate,  in  this  organism, 
either  the  "Filbrillenschicht"  or  the  transverse  "Fibrillen."  Ah  ex- 
amination of  the  microphotographs  (pis.  6,  7,  figs.  11-33)  will  show 
that  although  in  many  of  these  sections  in  which,  even  in  the  prints, 
the  separate  granules  of  the  macronucleus  (pi.  7,  figs.  27-29),  the 
separate  cilia  in  the  membranelles  (pi.  6,  figs.  14,  15,  19 ;  pi.  7,  fig. 
25),  and  the  individual  bacteria  within  the  food  vacuoles  (pi.  7,  fig. 
33)  may  be  fairly  well  made  out  (and  certain  it  is  that  all  details  may 
be  seen  to  much  better  advantage  in  the  original  sections)  there  is  no 
evidence  of  the  presence  of  these  above  described  "Fibrillen."  The 
fine  parallel  lines  of  the  lower  ends  of  figures  12-14,  plate  6,  might  at 
the  first  glance  be  confused  with  the  "  Fibrillenschicht "  of  Braune, 
but  on  a  closer  study  it  will  be  noted  that  these  lines  are  surface  mark- 
ings (cf.  fig.  11).  Also  an  examination  of  figures  26  to  32  will  show 
that  in  the  case  of  D.  ecaudatum  the  layer  separating  the  internal 
entoplasm  from  the  more  external  layers  (ectoplasm  and  cuticle)  is  a 
continuous,  homogeneous  membrane  rather  than  a  layer  of  "Fibril- 
len," as  pictured  and  described  by  Braune  for  0.  purkynjei,  with  the 
possible  exception,  as  noted  in  the  main  body  of  my  paper,  of  the 
extreme  posterior  end  of  the  body  in  which  the  oesophageal  wall  lies 
so  close  to  the  boundary  layer  as  to  defy  a  microscopic  separation  and 
identification  of  the  two  layers. 

3.  In  his  description  of  the  lips  of  the  dorsal  membranelle  zone 
(p.  157),  he  says,  "In  den  Wanden  aiiBern  Saumes  trifft  man  regel- 


1914]  Sharp:  Diplodinium  ecaudatum  107 

maBig  einzelne  starkere  quere  Fibrillen  (Fig.  37a)."  Evidently  there 
is  some  mistake  here  for  in  "Fig.  37"  the  "a"  refers  to  the  inner  lip 
and  apparently  the  walls  in  this  region  are  structurally  the  same  as  the 
walls  in  practically  all  other  parts  of  the  body.  And  in  speaking  of  the 
double  fastenings  of  the  dorsal  membranelles  (p.  158)  he  notes,  that, 
after  penetrating  the  outer  layer,  their  inner  ends  are  connected  with 
the  "  Fibrillenschicht "  by  short  inner  supports,  while  to  the  outer 
boundary  layer  decidedly  longer  "Fibrillen"  proceed  (Figs.  36,  37, 
In.  st.:  aus.  st.).  And  to  quote  from  his  description  of  the  outer  adoral 
lip  (pp.  158-159),  "Die  sie  umgebenden  Wiilste  zeigen  noch  starkere 
Fibrilleneinlagerung  wie  die  des  queren  Membranellenzugs. ' '  He  says 
further,  the  insertions  of  adoral  membranelles  are  similar  to  those  of 
the  dorsal  membranelles,  viz.,  the  double  fastening,  to  the  "Fibrillen- 
schicht" internally  and  to  the  "Grenzschicht"  externally.  Then  fol- 
lows a  detailed  description  of  a  very  complicated  fibrillar  system 
which  may  be  rather  briefly  summed  up  as  follows :  These  Stiitzfibril- 
len,"  as  Braune  terms  the  ciliary  roots,  which  here  lie  in  the  so-called 
"Entoplasma  a,"  are  extraordinarily  lengthened  out.  The  innermost 
set  of  these  parallel  "Fibrillen"  (Fig.  39,  St.  fbr.)  extend  anteriorly 
beyond  the  "  Stiitzapparat "  and  in  consequence  of  its  spine-like  pro- 
longations are  brought  together  in  a  circle.  Near  to  the  mouth  opening 
these  fibrillae  are  in  turn  encircled  by  a  " Fibrillenschlundring  (Fig. 
39,  Schlr.)."  On  their  oral  ends  are  imposed  the  oral  cilia  and  there- 
fore they  may  be  counted  as  "inneren  Stiitzen"  of  the  oral  mem- 
branelles. The  outer  supports  (AuBenstiitzen)  of  these  membranelles 
likewise  unite  by  threes  or  fours  into  "  Stiitzfibrillen "  which  again 
combine  to  form  larger  groups.  Each  of  these  larger  groups  has  only 
one  point  of  attachment,  which  is  found  either  in  the  "Stiitzapparat" 
itself  or  upon  the  two  "  Fibrillenstammen "  situated  just  above  the 
dorsal  membranelle  zone  (Fig.  39,  v  auf  &). 

This  double  fastening  of  the  membranelles,  viz.,  anterior  and  pos- 
terior roots  in  the  case  of  the  dorsal  membranelles  and  internal  and 
external  roots  in  the  case  of  the  adoral  membranelles,  has  been  de- 
scribed for  D.  ecaudatum  in  the  main  body  of  my  paper,  but  these 
fastenings  were  regarded  by  me  as  ciliary  root-filaments  rather  than 
as  supporting  fibrillae.  An  examination  of  figures  33  and  23  to  29 
will  show  that  in  D.  ecaudatum  the  only  fibrillae  present  in  the  adoral 
region  are  those  which  are  imbedded  in  the  oesophageal  walls,  and 
according  to  Braune 's  description  such  a  picture  as  is  obtained  in  the 
contracted  forms  (text  fig.  D)  would  be  absolutely  impossible. 


108  University  of  California  Publications  in  Zoology        [VOL.  13 

4.  A  rich  fibrillar  supply  is  also  described  for  the  walls  of  the  rec- 
tum and  for  the  region  of  the  bases  of  the  spines.  One  or  more  of 
these  fibrillae  are  described  as  extending  down  into  each  spine  as  a 
sort  of  axial  rod. 

A  fibrillar  layer  is  present  in  the  internal  wall  of  the  rectal  sheath 
of  Diplodinium  ecaudatum,  as  has  been  described  in  the  body  of  my 
paper,  but  as  was  there  pointed  out,  these  rectal  fibrillae  (rect.  f.,  pi. 
3,  fig.  3),  are,  in  the  ontogeny  of  D.  ecaudatum,  derived  from  the 
ventral  edge  of  the  preexisting  oesophageal  wall  and  are  not  exactly 
comparable  to  the  "quere  Fibrillenziige  "  described  for  the  correspond- 
ing region  in  Ophryoscolex  purkynjei.  These  rectal  fibers,  however, 
as  well  as  those  fibers  in  that  portion  of  the  oesophageal  wall  which  I 
have  described  as  lying  so  close  to  the  boundary  layer,  in  the  posterior 
end  of  the  animal,  as  to  defy  microscopic  separation  from  the  boundary 
layer,  are  undoubtedly  the  homologues  of  the  "Fibrillen"  described 
by  Braune  for  the  corresponding  region  in  0.  Purkynjei.  Other  than 
this  no  general  fibrillation  corresponding  to  that  described  for  0.  Pur- 
kynjei has  been  discovered  in  the  forms  investigated  by  me. 

In  regard  to  the  function  of  this  "Fibrillenreichtum,"  Braune 
concludes,  "daB  die  Fibrillen  keine  Myoneme,  sondern  einfache  elas- 
tische  Stabchen  sind,  denen  nur  die  Stiitzfunktion  zukommt."  And 
further  he  suggests  that  the  retraction  of  the  peristome  and  the  mem- 
branelle  zones  is  easily  brought  into  harmony  with  his  view.  To  quote 
again  (p.  161),  "Das  Protoplasma  bleibt  nach  wie  vor  ein  scheinbar 
homogenes,  zahlfliissiges  Medium,  daB  bei  unseren  Formen  durch  die 
Fibrillenanhaufungen  an  eine  starre  Gestalt  gebunden  wird.  Ver- 
moge  der  Elastizitat  der  einzelnen  Fibrillen  ist  das  Protoplasma  im- 
stande,  irgendwelche  Reize  durch  schwache  Lageveranderung  der 
beiden  Wimperzonen  zu  beantworten. 

Aus  dem  Gesagten  geht  hervor,  dass  die  bi- 
zarre Korpergestalt  der  Ophryoscoleciden  nur 
durch  das  Fibrillensystem  erhalten  wird.' 

The  arguments  against  the  neuromotor  apparatus  of  D.  ecaudatum 
•serving  merely  as  a  supporting  structure,  the  interpretation  which 
Braune  places  upon  the  "Fibrillenapparat"  of  0.  purkynjei,  have  been 
given  rather  fully  in  my  paper.  It  is  unfortunate  indeed  that  no 
microphotographs  accompany  Braune 's  article  and  that  his  drawings 
are  so  fragmentary.  As  neither  his  description  nor  his  figures  give 
any  evidence  that  he  saw  the  motor  mass  which  shows  so  clearly  in 
microphotographs  of  D.  ecaudatum  (pi.  6,  figs.  15,  16;  pi.  7,  figs,  22, 
23,  33),  it  would  seem  either  that  a  homologous  structure  is  lacking  in 


Sharp:  Diplodinium  ecaudatum  109 

0.  purkynjei  or  that  it  was  overlooked  by  the  observer.  Also,  as  has 
just  been  noted,  such  fibrillar  structures  as  the  "  Fibrillenschict, " 
"  Querfibrillen, "  "  Stiitzfibrillen, "  "queren  Korperfibrillen, "  and  the 
"Vereinigung  der  AuBen  stiitzen  des  inneren  kleinen  Bogens, "  and 
"des  auBeren  groBen  Bogens"  described  for  0.  purkynjei  are  lacking  in 
D.  ecaudatum.  And  even  if  they  did  occur  as  figured  by  the  above 
named  author  it  would  be  hard  to  interpret  them  as  being  merely  sup- 
porting structures,  for  according  to  Braune's  figures  (pi.  6,  figs.  36- 
43)  these  "Fibrillen"  occur  most  abundantly,  first  in  those  regions 
of  the  body  which  are  otherwise  well  supported  by  the  definite  and 
ample  skeletal  structures,  and  secondly  in  that  region  which  is  the 
most  markedly  retractile,  viz.,  the  oesophageal  region.  In  the  former 
case  these  fibrillae,  in  the  role  of  supporting  structures,  would  be  super- 
fluous while  in  the  latter  case  it  would  seem  as  though  a  contractile 
function  might  be  more  logically  assumed  for  them. 

In  regard  to  the  role  played  by  the  spines  Braune  suggests  that 
by  means  of  these,  the  ciliates  which  he  considers  to  be  normally 
boring  rather  than  swimming  animals,  are  enabled  to  keep  from  slip- 
ping back  while  forcing  their  way  through  the  more  solid  masses  of 
food. 

Attention  has  already  been  called  to  the  fact  that  in  D.  ecaudatum 
the  spines,  when  present,  curve  in  towards  the  central  axis  of  the 
body.  This  seems  to  be  true  for  0.  purkynjei  as  well.  Such  being  the 
case  it  is  difficult  to  imagine  how  these  spines  may  be  of  any  assistance 
whatever  in  serving  to  keep  the  animal  from  slipping  back  through  the 
mass  of  food  particles  and  especially  would  this  be  so,  if,  as  Braune 
points  out  for  0.  purkynjei,  that  portion  of  the  body  just  anterior  to 
the  spines  is  greater  in  diameter  than  that  portion  upon  which  the 
spines  are  situated. 

Also  in  looking  at  the  matter  from  the  viewpoint  of  evolution, 
according  to  Braune's  interpretation  the  presence  of  these  spines  being 
of  advantage  to  the  animal,  it  might  be  expected  that  those  species  or 
forms  so  provided  with  posterior  spines  would  contain  the  greater 
number  of  individuals.  Such,  however,  is  not  the  case,  at  least  this  does 
not  hold  true  for  D.  ecaudatum.  Also  it  must  be  noted  that  many  of 
the  forms  which  are  provided  with  posterior  spines  are  flat,  do  not 
habitually  rotate  about  the  longitudinal  axis  and  are  essentially  free 
swimming  and  not  boring  forms. 

Zoological  Laboratory,  University  of  California. 
Transmitted  February,  1914. 


110  University  of  California  Publications  in  Zoology        [VOL.  13 

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tiere,   sowie    die    durch    erstere    veranlassten    Krankheiten,    deren 
Behandlung  und  Verhiitung.  II.   Teil:   pflanzliche  Parasiten   (Wei- 
mar, Voigt),  2  Aufl.,  xvi  -(-  xvi  +  837,  4  pis.,  2  figs,  in  text. 
WEISS,  D.  C.  F.  H. 

1869.     Specielle  Physiologie  der  Haussaugethiere  fur  Thierarzte  und  Land- 
wirthe  (Stuttgart,  Metzler),  xii  +  548,  80  figs,  in  text. 


EXPLANATION  OF  PLATES 

PLATE  3 

Diplodinium  ecaudatum  forma  ecaudatum  Fiorentini 
(Camera  lucida  drawings) 

Fig.  1.  View  of  right  side  of  body,  showing  surface  markings,  underlying 
structures  shown  in  outline.  X  1400. 

Fig.  2.  View  of  left  side  of  body,  showing  surface  markings,  underlying 
structures  shown  in  outline.  Same  animal  as  figure  1,  viewed  from  opposite  side. 
X  1400. 

ABBREVIATIONS 

ador.  m.  s. — adoral  membranelle  zone. 
ant.  c.  v. —  anterior  contractile  vacuole. 
D. — dorsal  surface  of  the  body. 
d.  in.  z. — dorsal  membranelle  zone. 
1.  sic.  a. — left  skeletal  area. 
mac. — macronucleus. 
mic. — micronucleus. 

post.  c.  v. — posterior  contractile  vacuole. 
r.  sk.  a. — right  skeletal  area. 
V. — ventral  surface  of  the  body. 
v.  sic.  a. — ventral  skeletal  area. 


[114] 


PLATE  4 

Diplodinium  ecaudatum  forma  ecaudatum  Fiorentini 

(Fixed  in  Zenker's  fluid  and  stained  with  modified  Mallory's  connective 
tissue  stain.  Camera  lucida  drawings,  colored  to  match  the  stain.) 

Fig.  3.  Median  sagittal  section  constructed  from  superimposed  camera  lucida 
drawings  from  three  sections,  each  5  microns  thick.  X  1500.  Compare  with 
microphotographs,  plate  6,  figures  12-18,  and  plate  7,  figure  33. 

Fig.  4.  Cross-section  through  region  of  dorsal  membranelle  zone  from  three 
sections,  each  6  microns  thick.  X  1500.  Compare  with  microphotographs,  plate 
7,  figures  23-25. 

Fig.  5.  Cross-section  (same  series  as  fig.  4)  through  the  micronueleus  (cf. 
fig.  3).  X  1500.  Compare  with  microphotograph,  plate  7,  figure  29. 


ABBREVIATIONS 


ador.  m. — adoral  membranelles.  In  figure 
3  the  leader  is  carried  beyond  the  mem- 
branelle. 

an. — anus. 

ant.  cil.  r. — anterior  ciliary  roots. 

ant.  c.  v. — anterior  contractile  vacuole. 

bd.  1. — boundary  layer  (ectoplasmic). 

dr.  oes.  r. — circumeosophageal  ring. 

caec. — caecum. 

cut. — cuticle. 

c.  v.  r. — region  about  contractile  vacuole. 
D. — dorsal  side  of  body. 

d.  disk — dorsal  disk. 

d.  fur. — dorsal  furrow. 

d.  m.  str. — dorsal  motor  strand. 

d.  m. — dorsal  membranelles. 

ect. — ectoplasm. 

ent. — entoplasm. 

fd.  vac. — food  vacuoles. 

i.  ador.  lip — inner  adoral  lip. 

i.  d.  lip — inner  dorsal  lip. 

L. — left  side  of  body. 

1.  sic.  a. — left  skeletal  area. 

mac. — macronucleus. 

mic. — micronueleus. 

m.  m. — motor  mass  (motorium). 


o.  ador.  fur. — outer  adoral  furrow. 

o.  ador.  lip — outer  adoral  lip. 

o.  d.  fur. — outer  dorsal  furrow. 

o.  d.  lip — outer  dorsal  lip. 

oes. — oesophagus  or  cytopharynx. 

oes.  f. — oesophageal  fibers. 

oes.  retr.  str. — oesophageal  rectrator 

strands. 

op. — operculum. 
op.  f. — opercular  fibers. 
or. — oral  opening,  mouth,  or  cytostome. 
or.  cil. — oral  cilia. 
or.  disk — oral  disk. 
•  post.  cil.  r. — posterior  ciliary  roots. 
post.  c.  v. — posterior  contractile  vacuole. 
JR. — right  side  of  body. 
rect. — rectum. 
red.  f. — rectal  fibers. 
r.  sk.  a. — right  skeletal  area. 
sk.  lam. — skeletal  laminae. 
susp.  f. — suspensory  fibers. 
V. — ventral  side  of  body. 
v.  and  r.  sk.  lam. — ventral  and  right 

skeletal  laminae. 
v.  sk.  a. — ventral  skeletal  area. 
11.  m. — nuclear  membrane. 


[116] 


PLATE  5 
(Camera  lucida  drawings  from  whole  mounts) 

Fig.  6.     Diplodinium  ecaudatum  forma  caudatum  Fiorentini.     From  left  side. 
X  700. 

Fig.  7.     Diplodinium  ecaudatum  forma  bicaudatum  forma  nova.     From  left 
side.     X  700. 

Fig.  8.     Diplodinium   ecaudatum  forma   tricaudatum   forma   nova.     Posterior 
one-fourth  of  body  from  left  side.     X  700. 

Fig.  9.     Diplodinium  ecaudatum   forma   quadricaudatum  forma   nova.      From 
left  side  of  body.     X  700. 

Fig.  10.     Diplodinium  ecaudatum  forma  cattanei.     From  right  side  of  body. 
X  700. 

ABBREVIATIONS 

sp.  1 — primary  spine. 
sp.  2 — secondary  spine. 
sp.  3 — tertiary  spine. 
sp.  4 — quaternary  spine. 
sp.  5 — quintary  spine. 


[118] 


UNIV.   CALIF.    PUBL.   ZOOL.   VOL.    13 


[SHARP]   PLATE  5 


Sp.  1. 


if 

w) 


Sp.  3. 
Sp.2. 
Sp.l. 

'8 


Sp.  1. 
Sp.2. 


Sp.4. 
Sp.3. 


Sp.3. 
Sp.2. 


\ 


Sp.4. 
Sp.5. 

Sp.  1. 

10 


PLATE  6 

Microphotographs.  Diplodinium  ecaudatum  forma  ecaudatum  fixed  in  Schau- 
dinn  's  alcoholic  sublimate  solution,  stained  in  Heidenhain  's  iron-haexmatoxylin. 
imbedded  in  paraffin,  and  sectioned  at  5/j..  Nine  sections  in  series.  X  600. 

Fig.  11;  Tangential  section,  right  side  of  the  anterior  one-half  of  animal 
nearer  to  the  ventral  than  to  the  dorsal  side;  shows  surface  markings  of  the 
right  skeletal  area. 

Fig.  12.  Note  the  anterior  end  of  the  macronucleus,  which  lies  close  to  the 
right  dorsal  wall  of  the  body. 

Fig.  13.  Shows  right  extremity  of  dorsal  membranelle  zone,  right  side  of 
adoral  membranelles,  outlines  of  oesophagus  and  micronuclens. 

Fig.  14.  Note  especially  the  cut  ends  of  the  opercular  fibers  (see  op.  f.,  pi. 
4,  fig.  3)  and  the  faint  outline  of  the  circumoesophageal  ring. 

Fig.  15.  Almost  a  median  sagittal  section  at  anterior  end,  but  comes  to  sur- 
face of  body  on  right  side  at  posterior  end.  Shows  dorsal  neuromotor  mass 
(motorium),  oesophageal  retractor  strands,  circumoesophageal  ring,  boundary 
layer  between  ectoplasm  and  entoplasm,  anterior  contractile  vacuole,  posterior 
limit  of  macronucleus,  and  ventral  skeletal  laminae. 

Fig.  16.  Shows  food  vacuoles  in  entoplasm,  ventral  skeletal  laminae,  faint 
outline  of  posterior  contractile  vacuole  between  entoplasm  and  ectoplasm  just 
dorsal  to  posterior  end  of  macronucleus;  and  at  the  same  level,  near  the  ventral 
wall,  may  be  seen  the  darkly  stained  granular  faecal  mass  filling  the  caecum. 

Fig.  17.     Note  posterior  contractile  vacuole. 

Fig.  18.     Shows  rectum  and  anus. 

Fig.  19.     Adoral  membranelles  and  rectum. 


[120] 


UNIV.   CALIF,    PUBL.   ZOOL,   VOL    13 


[SHARP]   PLATE  6 


ie 


17 


19 


PLATE  7 

Figs.  20-32.  Cross-sections  of  Diplodinium  ecaudatum,  fixed  in  Zenker's 
fluid,  stained  in  modified  Mallory's  connective  tissue  stain,  embedded  in  paraf- 
fin, and  sectioned  at  6  microns.  Microphotographs.  X  750.  This  series  (series 
A-2-c-l)  contains  twenty  sections,  of  which  only  eleven  are  here  reproduced, 
figure  20  being  the  second  section  of  the  series  and  figure  32  the  nineteenth 
section;  figures  29  and  30  are  taken  from  a  second  series  (series  A-2-C-2). 
Section  1,  although  clear  on  the  slide,  could  not  be  satisfactorily  photographed. 

Fig.  20.  Section  2  of  series  A-2-c-l  is  cut  somewhat  obliquely  with  refer- 
ence to  the  longitudinal  axis  of  the  body,  but  in  an  almost  exact  transverse 
plane  with  reference  to  the  oral  and  adoral  region  of  the  animal.  Note  the 
oral  opening  (cf.  fig.  A)  and  adoral  membranelles. 

Fig.  21.  Section  3,  series  A-2-c-l.  Although  cut  more  deeply  than  figure 
20,  this  section  shows  the  oral  and  adoral  arrangement  to  even  better  advan- 
tage (cf.  figs.  A,  C). 

Pig.  22.     Shows  the  left  extremity  of  the  adoral  row  of  membranelles. 

Figs.  23-25.  Sections  5-7,  series  A-2-c-l.  Taken  through  the  dorsal  mem- 
branelle  zone.  Compare  with  plate  4,  figure  4.  Note  especially  the  darkly 
stained  oesophageal  fibers  (cf.  oes.  f.,  pi.  4,  fig.  4)  and  the  dorsal  motor  strand 
(cf.  d.  TO.  sir.,  pi.  4,  fig.  4). 

Fig.  26.  Note  outer  and  inner  dorsal  lips  (cf.  o.  d.  lip  and  i.  d.  lip,  pi.  4, 
tig.  4)  and  the  three  skeletal  regions. 

Fig.  27.  Section  9,  series  A-2-c-l.  Shows  anterior  end  of  macronucleus 
and  areolar,  contractile  vacuole  region  (cf.  c.  v.  r.,  pi.  4,  fig.  3). 

Fig.  28.     Shows  especially  well  the  anterior  contractile  vacuole. 

Fig.  29.  Section  11,  series  A-2-C-2.  This  series  is  from  a  somewhat  larger 
animal  and  was  taken  from  an  ox  which  had  not  been  fed  for  some  18  hours. 
Note  that  there  are  no  food  vacuoles  in  the  entoplasm.  Shows  macronucleus 
and  micronucleus  and  proximity  of  oesophageal  wall  to  the  micronucleus  (cf. 
pi.  4,  fig.  4). 

Fig.  30.     Section  15,  series  A-2-C-2.     Shows  posterior  contractile  vacuole. 

Fig.  31.  Section  17,  series  A-2-c-l.  Through  posterior  extremity  of  macro- 
nucleus  and  posterior  contractile  vacuole,  and  through  junction  of  rectum  and 
caecum. 

Fig.  32.     Section  19,  series  A-2-c-l.     Shows  rectum  enlarging  into  anal  slit. 

Fig.  33.  Paramedian  section  of  D.  ecaudatum  forma  ecaudatum,  just  to  right 
of  median  sagittal  plane.  Microphotograph  of  15  y.  section.  X  600.  (Cf.  pi.  4, 
fig.  3.) 


[122] 


UNIV.   CALIF.    PUBL.   ZOOL   VOL.    13 


[SHARP]   PLATE  7 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS— (Continued) 

6.  On  the  Skeletal  Morphology  of  Gonyaulax  r.aienata   (Levander),  by 
Charles  Atwood  Sofoid,    Pp.  287-294,  plate  18. 

6.  Dinoflagellata  of  the  San  Diego  Eegion,  V.  On  Spiraulax,  a  New  Genus 

of  the  Peridinida,  by  Charles  Atwood  Kofoid.    Pp.  295-300,  plate  19. 

Nos.  4,  5,  and  6  in  one  cover.    September,  1911  IJHJ 

7.  Notes  on  Some  Cephalopods  in  the  Collection  of  the  University  of  Cali- 

fornia, by  S.  S.  Berry.    Pp.  301-310,  plates  20-21.    September,  1911.      .10 

8.  On  a  Self -Closing  Plankton  Net  for  Horizontal  Towing,  by  Charles 

Atwood  Kofoid.    Pp.  311-348,  plates  22-25. 

9.  On  an  Improved  Form  of  Self-closing  Water-bucket  for  Planktoa  In- 

vestigations, by  Charles  Atwood  Kofoid.    Pp.  349-352. 

Nos.  8  and  9  in  one  cover.    November,  1911  _.      M 

Index,  pp.  353-357. 

Vol.  9.  1.  The  Horned  Lizards  of  California  and  Nevada  of  the  Genera  Phryno- 
soma  a«d  Anota,  by  Harold  C.  Bryant.  Pp.  1-84,  plates  1-9.  Decem- 
ber, 1911  70 

2.  On  a  Lyinphoid  Structure  Lying  Over  the  Myelencephalon  of  Lepisos- 

teus,  by  Asa  C.  Chandler.    Pp.  85-104,  plates  10-12.    December,  1911.      .25 

5.  Studies  on  Early  Stages  of  Development  in  Eats  and  Mice,  No.  3,  by 

E.  L.  Mark  and  J.  A.  Long.  The  Living  Eggs  of  Eats  and  Mice  with 
a  Description  of  Apparatus  for  Obtaining  and  Observing  Them  (Pre- 
liminary paper),  by  J.  A.  Long.  Pp.  105-136,  plates  13-17.  February, 
1912 30 

4.  The  Marine  Biological  Station  of  San  Diego,  Its  History,  Present  Con- 

ditions, Achievements,  and  Aims,  by  Wm.  E.  Sitter,  Pp.  137-248, 
plates  18-24,  and  2  maps.  March,  1912  _ l.Ofl 

6.  Oxygen  and  Polarity  in  Tubularia,  by  Harry  Beal  Torrey.    Pp.  249- 

251.     May,  1912  OS 

6.  The  Occurrence  and  Vertical  Distribution  of  the  Copepod*  of  the  San 

Diego  Eegion,  with  particular  reference  to  Nineteen  Species,  by  Cal- 
vin O.  Esterly.  Pp.  253-340,  7  text-figures.  July,  1912  1.0Q 

7.  Observations  on  the  Suckling  Period  in  the  Guinea-Pig,  by  J.  Marlon 

Eead.    Pp.  341-351.    September,  1912  _      .10 

8.  Haeokel's  Sethocephnlus  eucecryphalus  (Eadiolaria),  a  Marine  Oiliate, 

by  Charles  Atwood  Kofoid.    Pp.  353-357.    September,  1912  .05 

Index,  pp.  359-365. 

VoL  10.     (Contributions  from  the  Museum  of  Vertebrate  Zoology.) 

1.  Eeport  on  a  Collection  of  Birds  and  Mammals  from  Vancouver  Island, 

by  Harry  S.  Swarth.    Pp.  1-124,  plates  1-4.    February,  1912  1.00 

2.  A  New  Cony  from  the  Vicinity  of  Mount  Whitney,  by  Joseph  GrinneU. 

Pp.  125-129.    January,  1912  05 

5.  The  Mole  of  Southern  California,  by  J.  Grinnell  and  H.  S,  Swarth. 

Pp.  131-136,  2  text-figures. 

4.  Myotis  orinomus  Elliott,  a  Bat  New  to  California,  by  J.  GrinneU  and 

H.  S.  Swarth.    Pp.  137-142,  2  text-figures. 

Nos.  3  and  4  in  one  cover.    April,  1912  12 

5.  The  Bighorn  of  the  Sierra  Nevada,  by  Joseph  Grinnell.    Pp.  143-153, 

4  text-figures.    May,  1912  10 

8.  A  New  Perognafhus  from  the  San  Joaquin  Valley,   California,  by 
Walter  P.  Taylor.    Pp.  155-166,  1  text-figure. 

7.  The  Beaver  of  West  Central  California,  by  Walter  P.  Taylor.    Pp. 

167-169. 

Nos.  6  and  7  in  one  cover.    May,  1912 15 

8.  The  Two  Pocket  Gophers  of  the  Eegion  Contiguous  to  the  Lower  Colo- 

rado Eiver,  in  California  and  Arizona,  by  Joseph  Grinnell.  Pp.  171- 
178.  June,  1912  15 

9.  The  Species  of  the  Mammalian  Genus  Sorcx  of  West-Central  Cali- 

fornia, with  a  note  on  the  Vertebrate  Palustrine  Faunas  of  the 

Eegion,  by  Joseph  Grinnell.  Pp.  179-195,  figs.  1-6.    March,  1913 15 

10.  An  Account  of  the  Birds  and  Mammals  of  the  San  Jacinto  Area  of 

Southern  California,  with  Remarks  Upon  the  Behavior  of  Geographic 
^       Races  on  the  Margins  of  Their  Habitats,  by  J.  Grinnell  and  H.  S. 

Swarth.    Pp.  197-406,  pis.  6-10.    October,  1913 2.00 

Index,  pp.  407-417. 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS— (Continued) 

Vol.  11.    1.  Birds  in  Relation  to  a  Grasshopper  Outbreak  in  California,  by  Harold 

C.  Bryant.    Pp.  1-20.    November,  1912  20 

2.  On  the  Structure  and  Relationships  of  Dinosphaera  palustris  (Lemm.), 

by  Charles  Atwood  Kofoid  and  Josephine  Rigden  Michener.    Pp.  21- 

28.     December,   1912  10 

3.  A   Study   of   Epithelioma   Contagiosum   of   the   Common   Fowl,    by 

Clifford  D.  Sweet.    Pp.  29-51.    January,  1913  25 

4.  The  Control  of  Pigment  Formation  in  Amphibian  Larvae,  by  Myrtle 

E.  Johnson.    Pp.  53-88,  plate  1.    March,  1913  85 

6.  Sayitta  calif  ornica,  n.  sp.,  from  the  San  Diego  Region,  including 
Remarks  on  Its  Variation  and  Distribution,  by  Ellis  L.  Michael. 
Pp.  89-126,  plate  2.  June,  1913  .35 

6.  Pycnogonida  from  the  Coast  of  California,  with  Description  of  Two 

New  Species,  by  H.  V.  M.  Hall.  Pp.  127-142,  plates  3-4.  August,  1913.      .20 

7.  Observations  on  Isolated  Living  Pigment  Cells  from  the  Larvae  of 

Amphibians   by  S.  J.  Holmes.    Pp.  143-154,  plates  5-6. 

8.  Behavior  of  Ectodermic  Epithelium  of  Tadpoles  when  Cultivated  IB 

Plasma,  by  S.  J.  Holmes.     Pp.  155-172,  plates  7-8. 

Nos.  7  and  8  in  one  cover.    September,  1913 30 

9.  On  Some  Californian  Schizopoda,  by  H.  J.  Hansen.    Pp.  173-180,  pi.  9. 

November,  1913 10 

10.  Fourth  laxonomic  Report  on  the  Copepoda  of  the  San  Diego  Region, 

by  Calvin  O.  Esterly.    Pp.  181-196,  pis.  10-12.    November,  1913 .15 

11.  The  Behavior  of  Leeches  with  Especial  Reference  to  Its  Modifiability, 

A.  The  General  Reactions  of  the  Leeches  Dina  microstoma  Moore  and 
Glossiphonia  stagnates  Linnaeus;  B.  Modifiability  in  the  Behavior  of 
the  Leech  Dina  microstoma  Moore,  by  Wilson  Gee.  Pp.  197-305,  13 
text  figures.  December,  1913 _.  1.00 

12.  The  Structure  of  the  Ocelli  of  Polyorchis  penicillata,  by  Etta  Viola 

Little.    Pp.  307-328,  plates  13-15.    February,  1914  20 

13.  Modifications  and  Adaptations  to  Functions  in  the  Feathers  of  Circus    , 

Jmdsonius,  by  Asa  C.  Chandler.  Pp.  329-376,  plates  16-20.  March, 
1914  50 

14.  A  Determination  of  the  Economic  Status  of  the  Western  Meadowlark 

(Sturnella  neglecta)  in  California,  by  Harold  Child  Bryant.    Pp.  377- 

510,  plates  21-24,  5  text  figures.    February,  1914  1.25 

15.  Parasynaptic  Stages  in  the  Testis  of  Aneides  lugubris  (Hallowell),  by 

Harry  James  Snook  and  J.  A.  Long.    Pp.  511-528,  plates  25-26,  1  text 

fig.     April,  1914  25 

Vol.  12.    1.  A  Study  of  a  Collection  of  Geese  of  the  Urania  canadensis  Group  from 
the  San  Joaquin  Valley,  California,  by  Harry  S.  Swarth.    Pp.  1-24, 

plates  1-2,  8  text  figs.    November,  1913 SO 

2.  Nocturnal  Wanderings  of  the  California  Pocket  Gopher,  by  Harold  C. 

Bryant.    Pp.  25-29,  1  text  fig.    November,  1913  05 

5.  The  Reptiles  of  the  San  Jacinto  Area  of  Southern  California,  by  Sarah 

Rogers  Atsatt.    Pp.  31-50.    November,  1913  .20 

4.  An  Account  of  the  Mammals  and  Birds  of  the  Lower  Colorado  Valley, 

with  Especial  Reference  to  the  Distributional  Problems  Presented, 

by  Joseph  Grinnell.  Pp.  51-294,  plates  3-13,  9  text  figs.   March,  1914.    2.40 

5.  Aplodontia  chryseola,  a  New  Mountain  Beaver  from  the  Trinity  Region 

of  Northern  California,  by  Louise  Kellogg.    Pp.  295-296. 

6.  A  Previously  Undescribed  Aplodontia  from  the  Middle  North  Coast  of 

California,  by  Walter  P.  Taylor.    Pp.  297-300. 

Nos.  5  and  6  in  one  cover.    April,  1914  05 

7.  A  Second  Species  of  the  Mammalian  Genus  Microdipodops  from  Cali- 

fornia, by  Joseph  Grinnell.    Pp.  301-304.    April,  1914  05 

Vol.  13.    1.  The  Schizopoda  of  the  San  Diego  Region,  by  Calvin  O.  Esterly.    Pp. 

1-20,  plates  1-2,    April,  1914  15 

2.  A  Study  of  the  Occurrence  and  Manner  of  Distribution  of  the  -Cteno- 

phora  of  the  San  Diego  Region,  by  Calvin  O.  Esterly.  Pp.  21-38. 
April,  1914  15 

3.  A  New  Self -Regulating  Paraffin  Bath,  by  C.  W.  Woodworth.    Pp.  39- 

42,  2  text-figures.     April,  1914  05 

4.  Diplodinium  ecaudatum,  with  an  Account  of  Its  Neuromotor  Apparatus, 

by  Robert  G.  Sharp.  Pp.  43-122,  plates  3-7,  4  text  figures.  May, 
1914 .80 

5.  The  Vertical  Distribution  and  Movements  of  the   Schizopoda  of  the 

San  Diego  Region,  by  Calvin  O.  Esterly.    Pp.  123-145.    May,  1914 20 


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